Ssx-2 peptides presented by hla class ii molecules

ABSTRACT

The invention describes HLA class II binding peptides encoded by the SSX-2 tumor associated gene, as well as nucleic acids encoding such peptides and antibodies relating thereto. The peptides stimulate the activity and proliferation of CD4 +  T lymphocytes. Methods and products also are provided for diagnosing and treating conditions characterized by expression of the SSX-2 gene.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/565,315, filed Apr. 16, 2006, which is a national stage filing under35. U.S.C. §371 of PCT international patent application PCT/US04/23544,filed Jul. 21, 2004, which was published under PCT Article 21(2) inEnglish, and which claims priority under 35 U.S.C. §119 to U.S.provisional patent application Ser. No. 60/489,257, filed Jul. 22, 2003,the entire contents of each of which are incorporated herein byreference.

FIELD OF THE INVENTION

This invention relates to fragments of the tumor associated gene productSSX-2 which bind to and are presented to T lymphocytes by HLA class IImolecules. The peptides, nucleic acid molecules which code for suchpeptides, as well as related antibodies and CD4⁺ T lymphocytes, areuseful, inter alia, in diagnostic and therapeutic contexts.

BACKGROUND OF THE INVENTION

The process by which the mammalian immune system recognizes and reactsto foreign or alien materials is complex. An important facet of thesystem is the T cell response, which in part comprises mature Tlymphocytes which are positive for either CD4 or CD8 cell surfaceproteins. T lymphocytes can recognize and interact with other cells viacell surface complexes of peptides and molecules referred to as humanleukocyte antigens (“HLAs”) or major histocompatibility complexes(“MHCs”). These peptides are derived from larger molecules which areprocessed by the cells which also present the HLA/MHC molecule. See Maleet al., Advanced Immunology (J.P. Lipincott Company, 1987), especiallychapters 6-10. The interaction of T cells and complexes of HLA/peptideis restricted, requiring a specific T cell for a specific complex of anHLA molecule and a peptide. If a specific T cell is not present, thereis no T cell response even if its partner complex is present. Similarly,there is no response if the specific complex is absent, but the T cellis present. The mechanisms described above are involved in the immunesystem's response to foreign materials, in autoimmune pathologies,cellular abnormalities, and in responses to cancer.

The ability of the T cell arm of the tumor immune response todistinguish tumor cells from normal tissues with exquisite specificity,provides the basis for the development of T cell based cancerimmunotherapy. This specific recognition is the result of thepreferential or exclusive expression of some antigens in tumors ascompared to normal tissues. Several categories of antigens with more orless tumor-restricted expression have been identified during the lastdecade. Most of them correspond to non mutated self-antigens with tissuerestricted expression, although tumor-specific mutated antigens havealso been identified (Robbins et al., J Exp Med, 1996, 183:1185-1192).Tissue-specific differentiation antigens such as Melan-A or gp100(Kawakami et al, J Exp Med, 1994, 180:347-352; Coulie, J Exp Med, 1994.180:35-42) expressed by both normal cells of the melanocytic lineage andmalignant melanoma cells, and often spontaneously immunogenic inmelanoma patients, have been extensively studied. The group of tumorantigens most relevant for the development of generic cancer vaccines,however, is that of the so-called cancer/testis antigens (CTA) (Scanlanet al. Immunol Rev 2002. 188:22-32), whose gene expression isdevelopmentally regulated, being mostly restricted to gametogenic cellsbut silent in adult normal cells. Possibly as the result of activationof a common gametogenic protein expression program in cancer cells (Oldet al. Cancer Immunity 2001. 1:1). CTA are expressed in variableproportions of tumors of different histological types.

Numerous MHC Class I restricted epitopes recognized by tumor reactiveCD8⁺ T cells and specific for antigens in each of the groups listedabove have been identified. Interestingly, spontaneous CD8⁺ T cellresponses directed against several of these epitopes have been detectedin cancer patients (Valmori et al, Cancer Res, 2000. 60:4499-4506;Valmori et al., Cancer Res, 2001, 61:501-512). In contrast, theidentification of MHC Class II restricted epitopes recognized by tumorantigen specific CD4⁺ T cells has proven to be more difficult possiblybecause of the relatively low frequency of the latter and/or to the lackof effective identification methods (Klenerman et al, Nat Rev Immunol,2002, 2:263-272). Lately, however, because of important technicaladvances, the identification of CD4⁺ T cell epitopes derived from tumorantigens including CTA has been reported with increasing frequency(Chaux et al. J Exp Med 1999. 189:767-778; Zeng et al. Proc Natl AcadSci USA 2001. 98:3964-3969).

Because most nonhematopoietic tumors express MHC Class I but not ClassII molecules, it has been assumed that the predominant antitumor T cellmediated effector mechanism in vivo is direct killing of tumor cells bytumor antigen specific CD8⁺ T lymphocytes (CTL). CTL can indeed directlyand efficiently lyse tumor cells resulting sometimes in in vivoregression of large tumor masses. It is, however, becoming increasinglyclear that both tumor antigen specific CD8⁺ and CD4⁺ T cell responsesare important for efficient tumor immune response to occur in vivo(Wang, Trends Immunol. 2001. 22:269-276).

The multiple roles that tumor antigen specific CD4⁺ T cells canpotentially play in mediating antitumor functions are beingprogressively unveiled. These involve different to mechanisms fromproviding help for both priming and maintenance of tumor antigenspecific CD8⁺ T cells, to activation of B cells for production of tumorantigen specific antibodies, and even including more direct effects inthe effector phase of tumor rejection. The identification of CD4⁺ T cellepitopes toward which spontaneous responses arise in cancer patients isof particular interest as it gives the opportunity to analyze suchresponses and their underlying molecular mechanisms in vivo.Furthermore, there exist many patients who would not benefit from anytherapy which includes helper T cell stimulation via the aforementionedpeptides. Accordingly, there is a need for the identification ofadditional tumor associated antigens which contain epitopes presented byMHC Class II molecules and recognized by CD4⁺ lymphocytes.

SUMMARY OF THE INVENTION

It now has been discovered that the SSX-2 gene (also known asHOM-MEL-40) encodes HLA class II binding peptides that are epitopespresented by HLA-DP. These peptides, when presented by an antigenpresenting cell having the appropriate HLA class II molecule,effectively induce the activation and proliferation of CD4⁺ Tlymphocytes.

The invention provides isolated SSX-2 peptides which bind HLA class IImolecules, and functional variants of such peptides. The functionalvariants contain one or more amino acid additions, substitutions ordeletions to the SSX-2 peptide sequence. The invention also providesisolated nucleic acid molecules encoding such peptides, expressionvectors containing those nucleic acid molecules, host cells transfectedwith those nucleic acid molecules, and antibodies to those peptides andcomplexes of the peptides and HLA class II antigen presenting molecules.T lymphocytes which recognize complexes of the peptides and HLA class IIantigen presenting molecules are also provided. Kits and vaccinecompositions containing the foregoing molecules additionally areprovided. The foregoing can be used in the diagnosis or treatment ofconditions characterized by the expression of SSX-2. As it is known thatthe members of the SSX family of polypeptides and nucleic acids sharesignificant sequence identity and functional homology (e.g., as tumorantigens and precursors), the invention also embraces HLA bindingpeptides of similar amino acid sequence derived from members of the SSXfamily other than SSX-2 (see, e.g., Table III). Therefore, it isunderstood that the disclosure contained herein of SSX-2 HLA class II tobinding peptides, compositions containing such peptides, and methods ofidentifying and using such peptides applies also to other members of theSSX tumor associated antigen family.

According to one aspect of the invention, isolated SSX-2 HLA classII-binding peptides are provided. The peptides include an amino acidsequence set forth as SEQ ID NO:8, or a functional variant thereofcomprising 1-5 amino acid substitutions. The HLA class II-bindingpeptide or functional variant does not include a full length SSXprotein, particularly a full length SSX-2 protein. In certainembodiments, the isolated includes comprises an amino acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7. Preferablythe isolated peptide consists of an amino acid sequence selected fromthe group consisting of SEQ ID NO:1, SEQ ID NO:5, and SEQ ID NO:6, mostpreferably SEQ ID NO:1. Preferred functional variants include SEQ IDNOs:40-42 and SEQ ID NOs:44-48, as shown in Table III, and fragmentsthereof that bind HLA class II molecules.

In further embodiments, the isolated peptide includes an endosomaltargeting signal, preferably including an endosomal targeting portion ofhuman invariant chain Ii.

In other embodiments, the isolated peptide is non-hydrolyzable.Preferred non-hydrolyzable peptides include peptides comprising D-aminoacids, peptides comprising a -psi[CH₂NH]-reduced amide peptide bond,peptides comprising a -psi[COCH₂]-ketomethylene peptide bond, peptidescomprising a -psi[CH(CN)NH]-(cyanomethylene)amino peptide bond, peptidescomprising a -psi[CH₂CH(OH)]-hydroxyethylene peptide bond, peptidescomprising a -psi[CH₂O]-peptide bond, and peptides comprising a-psi[CH₂S]-thiomethylene peptide bond.

According to another aspect of the invention, compositions are providedthat include an isolated HLA class I-binding peptide and an isolatedSSX-2 HLA class II-binding peptide. The isolated SSX-2 HLA classII-binding peptide includes an amino acid sequence set forth as SEQ IDNO:8, or a functional variant thereof comprising 1-5 amino acidsubstitutions (but not including the full length of a SSX protein,particularly a full length SSX-2 protein). Preferably the HLA classI-binding peptide and the SSX-2 HLA class II-binding peptide arecombined as a polytope polypeptide.

In preferred embodiments, the isolated SSX-2 HLA class II-bindingpeptide includes to an amino acid sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6 and SEQ ID NO:7; most preferably the isolated SSX-2HLA class II-binding peptide consists of the amino acid sequence setforth as SEQ ID NO:1. Preferred functional variants include SEQ IDNOs:40-42 and SEQ ID NOs:44-48, as shown in Table III, and fragmentsthereof that bind HLA class II molecules.

In further embodiments, the isolated SSX-2 HLA class II-binding peptideincludes an endosomal targeting signal, preferably including anendosomal targeting portion of human invariant chain Ii.

According to a further aspect of the invention, compositions includingone or more of the foregoing isolated SSX-2 HLA class II-bindingpeptides complexed with one or more isolated HLA class II molecules areprovided. Preferably the number of isolated SSX-2 HLA class II-bindingpeptides and the number of isolated HLA class II molecules are equal.More preferably, the isolated SSX-2 HLA class II-binding peptides andthe isolated HLA class II molecules are coupled as a tetrameric moleculeof individual isolated SSX-2 HLA class II-binding peptides bound toindividual isolated HLA class II molecules. Even more preferably, theHLA class II molecules are DP molecules.

According to still another aspect of the invention, isolated nucleicacid molecules are provided that encode the foregoing SSX-2 HLA classII-binding peptides, provided that the nucleic acid molecule does notencode a full length SSX protein, particularly a full length SSX-2protein. Also provided are expression vectors including these isolatednucleic acid molecules operably linked to a promoter. In certainembodiments, the nucleic acid molecule includes a nucleotide sequenceselected from the group consisting of SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18 SEQ ID NO:19 SEQ ID NO:20 and SEQ ID NO:21,preferably SEQ ID NO:15. The foregoing expression vectors, in otherembodiments, also include a nucleic acid molecule that encodes an HLA-DPmolecule. Host cells transfected or transformed with the foregoingexpression vectors also are provided; in some embodiments, the host cellexpresses an HLA-DP molecule.

In another aspect of the invention, methods for selectively enriching apopulation of T lymphocytes with CD4⁺ T lymphocytes specific for a SSX-2HLA class II-binding peptide are provided. The methods includecontacting an isolated population of T lymphocytes with an to agentpresenting a complex of the SSX-2 HLA class II-binding peptide and anHLA class II molecule in an amount sufficient to selectively enrich theisolated population of T lymphocytes with the CD4⁺ T lymphocytes.

According to another aspect of the invention, methods for diagnosing acancer characterized by expression of SSX-2 HLA class II-binding peptideare provided. The methods include contacting a biological sampleisolated from a subject with an agent that is specific for the SSX-2 HLAclass II-binding peptide, and determining the interaction between theagent and the SSX-2 HLA class II-binding peptide as a determination ofthe disorder. Preferably the agent is an antibody or an antigen bindingfragment thereof.

According to yet another aspect of the invention, methods for diagnosinga cancer characterized by expression of a SSX-2 HLA class II-bindingpeptide which forms a complex with an HLA class II molecule areprovided. The methods include contacting a biological sample isolatedfrom a subject with an agent that binds the complex, and determiningbinding between the complex and the agent as a determination of thedisorder.

In still a further aspect of the invention, methods for treating asubject having a cancer characterized by expression of SSX-2 HLA classII-binding peptide are provided. The methods include administering tothe subject an amount of a SSX-2 HLA class II-binding peptide effectiveto ameliorate the disorder.

According to a further aspect of the invention, additional methods fortreating a subject having a cancer characterized by expression of SSX-2HLA class II-binding peptide are provided. These methods includeadministering to the subject an amount of a HLA class I-binding peptideand an amount of a SSX-2 HLA class II-binding peptide effective toameliorate the disorder. In some preferred embodiments, the HLA classI-binding peptide and the SSX-2 HLA class II-binding peptide arecombined as a polytope polypeptide. Preferably the HLA class I-bindingpeptide is a SSX-2 HLA class I-binding peptide.

According to another aspect of the invention, methods for treating asubject having a cancer characterized by expression of SSX-2 areprovided. The methods include administering to the subject an amount ofa SSX-2 HLA class II-binding peptide effective to ameliorate the cancer.

In another aspect of the invention, methods are provided for treating asubject having a cancer characterized by expression of SSX-2 HLA classII-binding peptide. The methods to include administering to the subjectan amount of autologous CD4⁺ T lymphocytes sufficient to ameliorate thedisorder, wherein the CD4⁺ T lymphocytes are specific for complexes ofan HLA class II molecule and a SSX-2 HLA class II-binding peptide.

In the foregoing methods, the SSX-2 HLA class II-binding peptidepreferably includes an amino acid sequence set forth as SEQ ID NO:8, ora functional variant thereof comprising 1-5 amino acid substitutions. Incertain preferred embodiments of the foregoing methods, the SSX-2 HLAclass II-binding peptide includes an amino acid sequence selected fromthe group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7. More preferably, theSSX-2 HLA class II-binding peptide consists of an amino acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:5 and SEQID NO:6, most preferably SEQ ID NO:1. Preferred functional variantsinclude SEQ ID NOs:40-42 and SEQ ID NOs:44-48, as shown in Table III,and fragments thereof that bind HLA class II molecules. In someembodiments, the HLA class II molecule is an HLA-DP molecule. In otherembodiments,the SSX-2 HLA class II binding peptide includes an endosomaltargeting signal, preferably an endosomal targeting portion of humaninvariant chain Ii.

In a further aspect of the invention, methods for identifying functionalvariants of a SSX-2 HLA class II-binding peptide are provided. Themethods include selecting a SSX-2 HLA class II-binding peptide whichincludes an amino acid sequence selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6 and SEQ ID NO:7, an HLA class II-binding molecule which binds theSSX-2 HLA class II-binding peptide, and a T cell which is stimulated bythe SSX-2 HLA class II-binding peptide presented by the HLA classII-binding molecule; mutating a first amino acid residue of the SSX-2HLA class II-binding peptide to prepare a variant peptide; anddetermining the binding of the variant peptide to HLA class II-bindingmolecule and the stimulation of the T cell. Binding of the variantpeptide to the HLA class II-binding molecule and stimulation of the Tcell by the variant peptide presented by the HLA class II-bindingmolecule indicates that the variant peptide is a functional variant.Exemplary functional variants that can be tested using such methods andused as controls in such methods include the preferred functionalvariants (SEQ ID NOs:40-42 and SEQ ID NOs:44-48, and fragments thereofthat bind HLA class II molecules).

In some embodiments, the methods include a step of comparing thestimulation of the T cell by the SSX-2 HLA class II-binding peptide andthe stimulation of the T cell by the functional variant as adetermination of the effectiveness of the stimulation of the T cell bythe functional variant.

According to another aspect of the invention, isolated polypeptides areprovided that bind selectively the foregoing SSX-2 HLA class II-bindingpeptides, provided that the isolated polypeptide is not an HLA class IImolecule. Also provided are isolated polypeptides that bind selectivelya complex of the foregoing SSX-2 HLA class II-binding peptides and anHLA class II molecule, provided that the isolated polypeptide is not a Tcell receptor. The foregoing isolated polypeptides preferably areantibodies, more preferably monoclonal antibodies. Preferred monoclonalantibodies include human antibodies, humanized antibodies, chimericantibodeis and single chain antibodies. In other embodiments, theisolated polypeptides are antibody fragments selected from the groupconsisting of Fab fragments, F(ab)₂ fragments, Fv fragments or fragmentsincluding a CDR3 region selective for a SSX-2 HLA class II-bindingpeptide.

The invention also provides isolated CD4⁺ T lymphocytes that selectivelybind a complex of an HLA class II molecule and a SSX-2 HLA classII-binding peptide, preferably wherein the HLA class II molecule is anHLA-DP molecule and wherein the SSX-2 HLA class II-binding peptideincludes an amino acid sequence set forth as SEQ ID NO:8 or a functionalvariant thereof. More preferably, the SSX-2 HLA class II-binding peptideincludes an amino acid sequence selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6 and SEQ ID NO:7. Still more preferably, the SSX-2 HLA classII-binding peptide consists of an amino acid sequence selected from thegroup consisting of SEQ ID NO:1, SEQ ID NO:5, and SEQ ID NO:6, mostpreferably SEQ ID NO:1. Preferred functional variants include SEQ IDNOs:40-42 and SEQ ID NOs:44-48, as shown in Table III, and fragmentsthereof that bind HLA class II molecules.

In a further aspect, the invention provides isolated antigen presentingcells that include a complex of an HLA class II molecule and a SSX-2 HLAclass II-binding peptide, preferably wherein the HLA class II moleculeis an HLA-DP molecule and wherein the SSX-2 HLA class II-binding peptidecomprises an amino acid sequence set forth as SEQ ID NO:8 or afunctional variant thereof. More preferably, the SSX-2 HLA classII-binding peptide includes to an amino acid sequence selected from thegroup consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7. Still more preferably, theSSX-2 HLA class II-binding peptide consists of an amino acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:5, and SEQID NO:6, most preferably SEQ ID NO:1. Preferred functional variantsinclude SEQ ID NOs:40-42 and SEQ ID NOs:44-48, as shown in Table III,and fragments thereof that bind HLA class II molecules.

According to another aspect of the invention, methods for identificationof HLA class II-binding epitopes of a protein are provided. The methodsinclude obtaining a peptide library of peptides that span the amino acidsequence of the protein; and contacting a population of cells containingCD4⁺ T lymphocytes with the peptide library in the presence of antigenpresenting cells to stimulate proliferation and/or cytokine productionby CD4⁺ T lymphocytes that selectively bind a peptide in the peptidelibrary. The stimulation of CD4⁺ T lymphocytes indicates that a peptidein the library contains at least one HLA class II epitope. In certainembodiments, the peptides are at least about 12 amino acids in length.In other embodiments, the peptides are between about 14 and about 50amino acids in length. Preferably the peptides are between about 20 andabout 22 amino acids in length.

In other embodiments, the peptides overlap each other by at least about4 amino acids, more preferably by at least about 10 amino acids.

In still other embodiments, the antigen presenting cells are autologousperipheral blood mononuclear cells.

The method can include additional steps of screening the isolated CD4⁺ Tlymphocytes with submixtures or single peptides, and/or clonallyexpanding the stimulated CD4⁺ T lymphocytes by periodic stimulation withthe selected peptide and/or isolating the stimulated CD4⁺ T lymphocytes.In the last case, it is preferred that the isolation of the stimulatedCD4⁺ T lymphocytes is carried out by cytokine guided flow cytometry cellsorting.

In some embodiments, the population of cells containing CD4⁺ Tlymphocytes also includes CD8⁺ T lymphocytes. In these embodiments, thestimulation of both CD4⁺ and CD8⁺ T lymphocytes indicates that a peptidein the synthetic library contains both HLA class I and HLA class IIepitopes.

The invention also provides pharmaceutical preparations containing anyone or more of the medicaments described above or throughout thespecification. Such pharmaceutical to preparations can includepharmaceutically acceptable diluents, carriers and/or excipients.

The use of the foregoing compositions, peptides, cells and nucleic acidsin the preparation of a medicament, particularly a medicament fortreatment of cancer, or for treating an immune response is alsoprovided.

These and other objects of the invention will be described in furtherdetail in connection with the detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows detection of SSX-2 specific CD4⁺ T cells in peptidestimulated cultures. The presence of specific CD4⁺ T cells in theculture from patient LAU 672 was assessed by intracellular staining withanti-IFN-γ (FIG. 1A, left panels) or anti-IL-2 antibodies (FIG. 1A,right panels) after stimulation with autologous PBMC alone (upperpanels) or with the peptide submixture P1-3 (containing SSX-2 peptides1-22, 13-34, 25-46). Numbers in upper right quadrants are percent ofcytokine producing cells among CD4⁺ T cells. The data obtained for allpeptide submixtures is shown in FIG. 1B.

FIG. 2 depicts determination of the minimal sequence optimallyrecognized by SSX-2 specific CD4⁺ T cells. Synthetic peptides extendedor truncated at the SSX-2₁₃₋₃₄ N- or C-terminus were used to determinethe minimal length of the epitope recognized by SSX-2 specific CD4⁺ Tcells. Serial dilutions of each peptide were incubated with EBV LAU 149and SSX-2 specific CD4⁺ T cells. IFN-γ secretion was determined by ELISAin the culture supernatant after 24 hrs of culture. The peptides testedwere:

(SSX-2₁₃_34; SEQ ID NO: 1) VGAQIPEKIQKAFDDIAKYFSK; (SSX-2₁₃_36; SEQ ID NO: 2) VGAQIPEKIQKAFDDIAKYFSKEE;(SSX-2₁₃_32; SEQ ID NO: 3) VGAQIPEKIQKAFDDIAKYF; (SSX-2₁₃_32; SEQ ID NO: 4)  VGAQIPEKIQKAFDDIAK;(SSX-2₁₇_34; SEQ ID NO: 5) IPEKIQKAFDDIAKYFSK; (SSX-2₁₉_34; SEQ ID NO: 6) EKIQKAFDDIAKYFSK;  (SSX-2₂₁_34; SEQ ID NO: 7)IQKAFDDIAKYFSK  and (SSX-2₆₁_82; SEQ ID NO: 30) LGFKATLPPFMCNKRAEDFQGN. 

FIG. 3 demonstrates recognition of SSX-2₁₃₋₃₄ by specific CD4⁺ T cellsin the context of HLA-DP. FIG. 3A: Intracellular IFN-γ secretion bySSX-2 specific CD4+ T cells (clone 3C8) upon stimulation with peptideSSX-213-34 was assessed both in the absence and in the presence of antiHLA-DR, -DP or -DQ antibodies. FIG. 3B: The ability of APC bearing todifferent HLA-DP alleles to present peptide SSX-2 13-34 to specific CD4⁺T cells was assessed by intracellular IFN-γ secretion.

FIG. 4 shows the lack of tumor recognition by SSX-2 13-34 specific CD4⁺T cells. FIG. 4A: Recognition of Me 260 cells by CD4+ T cells was testedby ELISPOT in the absence or in the presence of exogeneously addedpeptide SSX-213-34. Where indicated, cells were treated with IFN-γduring 48 hr. FIG. 4B: Recognition of T465A was assessed as in FIG. 3Aas well as upon transfection with an SSX-2 encoding plasmid. The CD8+ Tcell clone B3.4 specific for peptide SSX-2 41-49 was used as an internalcontrol.

FIG. 5 shows that the SSX-2 epitope recognized by 13-34 specific CD4⁺ Tcells is efficiently processed and presented by professional APC. FIG.5A: The ability of EBV LAU 149 to process the SSX-2 protein and presentthe relevant epitope to specific CD4+ T cells was assessed by ELISPOTafter 12 hrs incubation with soluble recombinant SSX-2 protein followedby washing. NY-ESO-1 protein was used as an internal control. FIGS. 5Band 5C: Processing and presentation of the SSX-2 CD4⁺ T cell epitope byLAU 672 autologous monocyte-derived DC was assessed as in FIG. 5A.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is a SSX-2₁₃₋₃₄ peptide (VGAQIPEKIQKAFDDIAKYFSK).

SEQ ID NO:2 is a SSX-2₁₃₋₃₆ peptide (VGAQIPEKIQKAFDDIAKYFSKEE).

SEQ ID NO:3 is a SSX-2₁₃₋₃₂ peptide (VGAQIPEKIQKAFDDIAKYF).

SEQ ID NO:4 is a SSX-2₁₃₋₃₂ peptide (VGAQIPEKIQKAFDDIAK).

SEQ ID NO:5 is a SSX-2₁₇₋₃₄ peptide (IPEKIQKAFDDIAKYFSK).

SEQ ID NO:6 is a SSX-2₁₉₋₃₄ peptide (EKIQKAFDDIAKYFSK).

SEQ ID NO:7 is a SSX-2₂₁₋₃₄ peptide (IQKAFDDIAKYFSK).

SEQ ID NO:8 is a SSX-2₁₉₋₃₀ peptide (EKIQKAFDDIAK).

SEQ ID NOs:9 and 10 are the nucleotide and amino acid sequences,respectively, for SSX-2 peptide isoform b/transcript variant 2 (nucleicacid=NM_(—)175698.1, GI:28559005; polypeptide=NP_(—)783629.1GI:28559006).

SEQ ID NOs:11 and 12 are the nucleotide and amino acid sequences,respectively, for SSX-2 peptide isoform a/transcript variant 1 (nucleicacid=NM_(—)003147.4, GI:28559004; polypeptide=NP_(—)003138.3GI:27659724).

SEQ ID NOs:13 and 14 are nucleotide and amino acid sequences,respectively, for a protein that is similar to SSX-2 isoformb/transcript variant 2 (nucleic acid=XM_(—)300501.1, GI:30157775;polypeptide=XP_(—)300501.1, GI:30157776).

SEQ ID NO:15 is a nucleotide sequence encoding the SSX-2 peptideSSX-2₁₃₋₃₄ (SEQ ID NO:1): gtt ggg gcc caa att ccc gaa aaa atc caa aaagcn ttt gat gat att gcn aaa tat ttt agt aaa.

SEQ ID NO:16 is a nucleotide sequence encoding the SSX-2 peptideSSX-2₁₃₋₃₆ (SEQ ID NO:2): gtt ggg gcc caa att ccc gaa aaa atc caa aaagcn ttt gat gat att gcn aaa tat ttt agt aaa gaa gaa).

SEQ ID NO:17 is a nucleotide sequence encoding the SSX-2 peptideSSX-2₁₃₋₃₂ (SEQ ID NO:3): gtt ggg gcc caa att ccc gaa aaa atc caa aaagcn ttt gat gat att gcn aaa tat ttt.

SEQ ID NO:18 is a nucleotide sequence encoding the SSX-2 peptideSSX-2₁₃₋₃₀ (SEQ ID NO:4): gtt ggg gcc caa att ccc gaa aaa atc caa aaagcn ttt gat gat att gcn aaa.

SEQ ID NO:19 is a nucleotide sequence encoding the SSX-2 peptideSSX-2₁₇₋₃₄ (SEQ ID NO:5): att ccc gaa aaa atc caa aaa gcn ttt gat gatatt gcn aaa tat ttt agt aaa.

SEQ ID NO:20 is a nucleotide sequence encoding the SSX-2 peptideSSX-2₁₉₋₃₄ (SEQ ID NO:6): gaa aaa atc caa aaa gcn ttt gat gat att gcnaaa tat ttt agt aaa.

SEQ ID NO:21 is a nucleotide sequence encoding the SSX-2 peptideSSX-2₂₁₋₃₄ (SEQ ID NO:7): gaa aaa atc caa aaa gcn ttt gat gat att gcnaaa tat ttt agt aaa.

SEQ ID NO:22 is the nucleotide sequence of the DPA forward primer (atgcgc cct gaa gac aga atg t).

SEQ ID NO:23 is the nucleotide sequence of the DPA reverse primer (tcacag ggt ccc ctg ggc ccg ggg ga).

SEQ ID NO:24 is the nucleotide sequence of the DPB forward primer (atgatg gtt ctg cag gtt tct g).

SEQ ID NO:25 is the nucleotide sequence of the DPB reverse primer (ttatgc aga tcc tcg ttg aac ttt c).

SEQ ID NO:26 is a SSX-2₁₋₂₂ peptide (MNGDDAFARRPTVGAQIPEKIQ).

SEQ ID NO:27 is a SSX-2₂₅₋₄₆ peptide (FDDIAKYFSKEEWEKMKASEKI).

SEQ ID NO:28 is a SSX-2₃₇₋₅₈ peptide (WEKMKASEKIFYVYMKRKYEAM).

SEQ ID NO:29 is a SSX-2₄₉₋₇₀ peptide (VYMKRKYEAMTKLGFKATLPPF).

SEQ ID NO:30 is a SSX-2₆₁₋₈₂ peptide (LGFKATLPPFMCNKRAEDFQGN).

SEQ ID NO:31 is a SSX-2₇₃₋₉₄ peptide (NKRAEDFQGNDLDNDPNRGNQV).

SEQ ID NO:32 is a SSX-2₈₇₋₁₀₅ peptide (DPNRGNQVERPQMTFGRLQ).

SEQ ID NO:33 is a SSX-2₉₇₋₁₁₈ peptide (PQMTFGRLQGISPKIMPKKPAE).

SEQ ID NO:34 is a SSX-2₁₀₉₋₁₃₀ peptide (PKIMPKKPAEEGNDSEEVPEAS).

SEQ ID NO:35 is a SSX-2₁₂₁₋₁₄₂ peptide (NDSEEVPEASGPQNDGKELCPP).

SEQ ID NO:36 is a SSX-2₁₃₃₋₁₅₄ peptide (QNDGKELCPPGKPTTSEKIHER).

SEQ ID NO:37 is a SSX-2₁₄₅₋₁₆₆ peptide (PTTSEKIHERSGPKRGEHAWTH).

SEQ ID NO:38 is a SSX-2₁₅₇₋₁₇₈ peptide (PKRGEHAWTHRLRERKQLVIYE).

SEQ ID NO:39 is a SSX-2₁₆₉₋₁₈₈ peptide (RERKQLVIYEEISDPEEDDE).

SEQ ID NO:40 is a SSX-1₁₃₋₃₄ peptide (DDAKASEKRSKAFDDIATYFSK).

SEQ ID NO:41 is a SSX-3₁₃₋₃₄ peptide (VGAQIPEKIQKAFDDIAKYFSK).

SEQ ID NO:42 is a SSX-4₁₃₋₃₄ peptide (DDAQISEKLRKAFDDIAKYFSK).

SEQ ID NO:43 is a SSX-5₁₃₋₃₄ (isoform a) peptide(VGSQIPEKMQKHPWRQVCDRGI).

SEQ ID NO:44 is a SSX-5₁₃₋₃₄ (isoform b) peptide(VGSQIPEKMQKAFDDIAKYFSE).

SEQ ID NO:45 is a SSX-6₁₃₋₃₄ peptide (DDAKASEKRSKAFDDIAKYFSK).

SEQ ID NO:46 is a SSX-7₁₃₋₃₄ peptide (AGAQIPEKIQKSFDDIAKYFSK).

SEQ ID NO:47 is a SSX-8₁₃₋₃₄ peptide (DDDKASEKRSKAFNDIATYFSK).

SEQ ID NO:48 is a SSX-9₁₃₋₃₄ peptide (AGSQIPEKIQKAFDDIAKYFSK).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides isolated SSX-2 peptides presented by HLA class IImolecules, which peptides stimulate the proliferation and activation ofCD4⁺ T lymphocytes. Such peptides are referred to herein as “SSX-2 HLAclass II binding peptides,” “HLA class II binding peptides” and “MHCclass II binding peptides.” Hence, one aspect of the invention is anisolated peptide which includes the amino acid sequence of SEQ ID NO:8,preferably any one of SEQ ID NOs: 1-7. The peptides referred to hereinas “SSX-2 HLA class II binding peptides” include fragments of SSX-2protein, but do not include full-length SSX-2 protein (e.g., SEQ IDNOs:10, 12 or 14). Likewise, nucleic acids that encode the “SSX-2 HLAclass to II binding peptides” include fragments of the SSX-2 gene codingregion, but do not include the full-length SSX-2 coding region (e.g., asfound in SEQ ID NOs: 9, 11 or 13).

The examples below show the isolation of peptides which are SSX-2 HLAclass II binding peptides. These exemplary peptides are processedtranslation products of an SSX-2 nucleic acid (e.g., SEQ ID NOs:9, 11and 13; the encoded polypeptide sequences are given as SEQ ID NOs:10, 12and 14). As such, it will be appreciated by one of ordinary skill in theart that the translation products from which a SSX-2 HLA class IIbinding peptide is processed to a final form for presentation may be ofany length or sequence so long as they encompass the SSX-2 HLA class IIbinding peptide. As demonstrated in the examples below, peptides orproteins as small as 14 amino acids and as large as the amino acidsequence of a SSX-2 protein (SEQ ID NOs:10, 12 and 14) are appropriatelyprocessed, presented by HLA class II molecules and effective instimulating CD4⁺ T lymphocytes. SSX-2 HLA class II binding peptides,such as the peptides of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7 may have one, two, three,four, five, six, seven, eight, nine, ten, 15, 20, 25, 30, 40, 50 or moreamino acids added to either or both ends. The antigenic portion of sucha peptide is cleaved out under physiological conditions for presentationby HLA class II molecules. It is also well known in the art that HLAclass II peptide length is variable between about 10 amino acids andabout 30 amino acids (Engelhard, Ann. Rev. Immunol. 12:181-201, 1994).Most of the HLA class II binding peptides fall in to the length range of12-19 amino acids. Nested sets of HLA class II binding peptides havebeen identified, wherein the peptides share a core sequence but havedifferent amino acids at amino and/or carboxyl terminal ends (see, e.g.,Chicz et al., J. Exp. Med. 178:27-47, 1993). Thus additional SSX-2 HLAclass II binding peptides comprising at least a portion of the sequencesof the peptides reported herein, preferably comprising SEQ ID NO:8, aswell as homologous SSX family HLA class II binding peptides (e.g., ofsimilar sequence from other SSX proteins including SSX-1, SSX-3 andSSX-4), can be identified by one of ordinary skill in the art accordingto the procedures described herein.

The procedures described in the Examples that were utilized to identifySSX-2 HLA class II binding peptides also can be utilized to identifyother HLA class II binding peptides, including homologous SSX family HLAclass II binding peptides. Thus, for example, one can load antigenpresenting cells, such as dendritic cells of normal blood donors, with ato recombinant SSX protein (or a number of overlapping peptide fragmentsthereof as is described herein) by contacting the cells with the SSXpolypeptide (or a series of peptides) or by introducing into the cells anucleic acid molecule which directs the expression of the SSX protein(or peptide) of interest. The antigen-presenting cells then can be usedto induce in vitro the activation and/or proliferation of specific CD4lymphocytes that recognize SSX HLA class II binding peptides. The CD4lymphocytes can be isolated according to standard methods, includingcytokine guided flow cytometry cell sorting as described herein. Thesequence of the peptide epitope then can be determined as described inthe Examples, e.g., by stimulating cells with peptide fragments of theSSX protein used to stimulate the activation and/or proliferation of CD4lymphocytes. If a peptide library is used in the initial screening, thensubsets of these peptides or individual peptides can be used for thesubsequent screening. Preferably the peptides are at least about 12amino acids in length for efficient binding to HLA class II molecules.More preferably, the peptides are between about 14 and about 50 aminoacids in length, still more preferably between about 20 and about 22amino acids in length. By using overlapping peptides, all possibleepitopes can be screened. In some embodiments, the peptides overlap eachother by at least about 4 amino acids, but preferably the peptidesoverlap each other by at least about 10 amino acids. In addition, onecan make predictions of peptide sequences derived from SSX familyproteins which are candidate HLA class II binding peptides based on theconsensus amino acid sequences for binding HLA class II molecules.Peptides which are thus selected can be used in the assays describedherein for inducing activation and/or proliferation of specific CD4lymphocytes and identification of peptides. Additional methods ofselecting and testing peptides for HLA class II binding are well knownin the art. The foregoing methods also can be used to simultaneouslyscreen a protein sequence for the presence of both HLA class I and HLAclass II epitopes by contacting the antigen presenting cells with apopulation of cells that contans both CD4⁺ T lymphocytes and CD8⁺ Tlymphocytes. The stimulation of both CD4⁺ and CD8⁺ T lymphocytesindicates that a peptide in the synthetic library contains both HLAclass I and HLA class II epitopes. Stimulation of CD8⁺ or CD4⁺ Tlymphocytes indicates that only HLA class I or HLA class II epitopesexist in a reactive peptide.

As noted above, the invention embraces functional variants of SSX-2 HLAclass II binding peptides. As used herein, a “functional variant” or“variant” of a HLA class II to binding peptide is a peptide whichcontains one or more modifications to the primary amino acid sequence ofa HLA class II binding peptide and retains the HLA class II and T cellreceptor binding properties disclosed herein. Modifications which createa SSX-2 HLA class II binding peptide functional variant can be made forexample 1) to enhance a property of a SSX-2 HLA class II bindingpeptide, such as peptide stability in an expression system or thestability of protein-protein binding such as HLA-peptide binding; 2) toprovide a novel activity or property to a SSX-2 HLA class II bindingpeptide, such as addition of an antigenic epitope or addition of adetectable moiety; or 3) to provide a different amino acid sequence thatproduces the same or similar T cell stimulatory properties.Modifications to SSX-2 (as well as SSX family) HLA class II bindingpeptides can be made to nucleic acids which encode the peptide, and caninclude deletions, point mutations, truncations, amino acidsubstitutions and additions of amino acids. Alternatively, modificationscan be made directly to the polypeptide, such as by cleavage, additionof a linker molecule, addition of a detectable moiety, such as biotin,addition of a fatty acid, substitution of one amino acid for another andthe like. Variants also can be selected from libraries of peptides,which can be random peptides or peptides based on the sequence of theSSX peptides including substitutions at one or more positions(preferably 1-5). For example, a peptide library can be used incompetition assays with complexes of SSX peptides bound to HLA class IImolecules (e.g. dendritic cells loaded with SSX peptide). Peptides whichcompete for binding of the SSX peptide to the HLA class II molecule canbe sequenced and used in other assays (e.g. CD4 lymphocyteproliferation) to determine suitability as SSX peptide functionalvariants. Preferred functional variants include SEQ ID NOs:40-42 and SEQID NOs:44-48, as shown in Table III, and fragments thereof that bind HLAclass II molecules.

Modifications also embrace fusion proteins comprising all or part of aSSX HLA class II binding peptide amino acid sequence, such as theinvariant chain-SSX-2 fusion proteins described herein. The inventionthus embraces fusion proteins comprising SSX-2 HLA class II bindingpeptides and endosomal targeting signals such as the human invariantchain (Ii). As is disclosed below, fusion of an endosomal targetingportion of the human invariant chain to SSX-2 resulted in efficienttargeting of SSX-2 to the HLA class II peptide presentation pathway. An“endosomal targeting portion” of the human invariant chain or othertargeting polypeptide is that portion of the molecule which, when fusedor conjugated to a second to polypeptide, increases endosomallocalization of the second polypeptide. Thus endosomal targetingportions can include the entire sequence or only a small portion of atargeting polypeptide such as human invariant chain Ii. One of ordinaryskill in the art can readily determine an endosomal targeting portion ofa targeting molecule.

Prior investigations (PCT/US99/21230) noted that fusion of an endosomaltargeting portion of LAMP-1 protein did not significantly increasetargeting of MAGE-A3 to the HLA class II peptide presentation pathway.It is possible that this was a MAGE-A3 specific effect. Therefore, theSSX-2 peptides of the invention can be tested as fusions with LAMP-1 todetermine if such fusion proteins are efficiently targeted to the HLAclass II peptide presentation pathway. Additional endosomal targetingsignals can be identified by one of ordinary skill in the art, fused toSSX-2 or a SSX-2 HLA class II binding portion thereof, and tested fortargeting to the HLA class II peptide presentation pathway using no morethan routine experimentation.

The amino acid sequence of SSX HLA class II binding peptides may be ofnatural or non-natural origin, that is, they may comprise a natural SSXHLA class II binding peptide molecule or may comprise a modifiedsequence as long as the amino acid sequence retains the ability tostimulate helper T cells when presented and retains the property ofbinding to an HLA class II molecule such as an HLA DP molecule. Forexample, SSX-2 HLA class II binding peptides in this context may befusion proteins including a SSX-2 HLA class II binding peptide andunrelated amino acid sequences, synthetic SSX-2 HLA class II bindingpeptides, labeled peptides, peptides isolated from patients with a SSX-2expressing cancer, peptides isolated from cultured cells which expressSSX-2, peptides coupled to nonpeptide molecules (for example in certaindrug delivery systems) and other molecules which include the amino acidsequence of SEQ ID NOs:1-8.

Preferably, the SSX-2 HLA class II binding peptides arenon-hydrolyzable. To provide such peptides, one may select SSX-2 HLAclass II binding peptides from a library of non-hydrolyzable peptides,such as peptides containing one or more D-amino acids or peptidescontaining one or more non-hydrolyzable peptide bonds linking aminoacids. Alternatively, one can select peptides which are optimal forinducing CD4⁺ T lymphocytes and then modify such peptides as necessaryto reduce the potential for hydrolysis by proteases. For example, todetermine the susceptibility to proteolytic cleavage, peptides may to belabeled and incubated with cell extracts or purified proteases and thenisolated to determine which peptide bonds are susceptible toproteolysis, e.g., by sequencing peptides and proteolytic fragments.Alternatively, potentially susceptible peptide bonds can be identifiedby comparing the amino acid sequence of a SSX-2 HLA class II bindingpeptide with the known cleavage site specificity of a panel ofproteases. Based on the results of such assays, individual peptide bondswhich are susceptible to proteolysis can be replaced withnon-hydrolyzable peptide bonds by in vitro synthesis of the peptide.

Many non-hydrolyzable peptide bonds are known in the art, along withprocedures for synthesis of peptides containing such bonds.Non-hydrolyzable bonds include, but are not limited to,-psi[CH₂NH]-reduced amide peptide bonds, -psi[COCH₂]-ketomethylenepeptide bonds, -psi[CH(CN)NH]-(cyanomethylene)amino peptide bonds,-psi[CH₂CH(OH)]-hydroxyethylene peptide bonds, -psi[CH₂O]-peptide bonds,and -psi[CH₂S]-thiomethylene peptide bonds.

Nonpeptide analogs of peptides, e.g., those which provide a stabilizedstructure or lessened biodegradation, are also contemplated. Peptidemimetic analogs can be prepared based on a selected SSX-2 HLA class IIbinding peptide by replacement of one or more residues by nonpeptidemoieties. Preferably, the nonpeptide moieties permit the peptide toretain its natural conformation, or stabilize a preferred, e.g.,bioactive, confirmation. Such peptides can be tested in molecular orcell-based binding assays to assess the effect of the substitution(s) onconformation and/or activity. One example of methods for preparation ofnonpeptide mimetic analogs from peptides is described in Nachman et al.,Regul. Pept. 57:359-370 (1995). Peptide as used herein embraces all ofthe foregoing.

If a variant involves a change to an amino acid of SEQ ID NOs:1-8,functional variants of the SSX-2 HLA class II binding peptide havingconservative amino acid substitutions typically will be preferred, i.e.,substitutions which retain a property of the original amino acid such ascharge, hydrophobicity, conformation, etc. Examples of conservativesubstitutions of amino acids include substitutions made amongst aminoacids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K,R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

Other methods for identifying functional variants of the SSX-2 HLA classII binding peptides are provided in a published PCT application ofStrominger and Wucherpfennig (PCT/US96/03182). These methods rely uponthe development of amino acid sequence motifs to which potentialepitopes may be compared. Each motif describes a finite set of aminoacid sequences in which the residues at each (relative) position may be(a) restricted to a single residue, (b) allowed to vary amongst arestricted set of residues, or (c) allowed to vary amongst all possibleresidues. For example, a motif might specify that the residue at a firstposition may be any one of the residues valine, leucine, isoleucine,methionine, or phenylalanine; that the residue at the second positionmust be histidine; that the residue at the third position may be anyamino acid residue; that the residue at the fourth position may be anyone of the residues valine, leucine, isoleucine, methionine,phenylalanine, tyrosine or tryptophan; and that the residue at the fifthposition must be lysine.

Other computational methods for selecting amino acid substitutions, suchas iterative computer structural modeling, can also be performed by oneof ordinary skill in the art to prepare variants. Sequence motifs forSSX-2 HLA class II binding peptide functional variants can be developedby analysis of the binding domains or binding pockets of majorhistocompatibility complex HLA-DP proteins and/or the T cell receptor(“TCR”) contact points of the SSX-2 HLA class II binding peptidesdisclosed herein. By providing a detailed structural analysis of theresidues involved in forming the HLA class II binding pockets, one isenabled to make predictions of sequence motifs for binding of SSXpeptides to any of the HLA class II proteins.

Using these sequence motifs as search, evaluation, or design criteria,one is enabled to identify classes of peptides (e.g. SSX HLA class IIbinding peptides, particularly the SSX-2 peptides disclosed herein, andfunctional variants thereof) which have a reasonable likelihood ofbinding to a particular HLA molecule and of interacting with a T cellreceptor to induce T cell response. These peptides can be synthesizedand tested for activity as described herein. Use of these motifs, asopposed to pure sequence homology (which excludes many peptides whichare antigenically similar but quite distinct in sequence) or sequencehomology with unlimited “conservative” substitutions (which admits manypeptides which differ at critical highly conserved sites), represents amethod by which one of ordinary skill in the art can evaluate peptidesfor potential application in the treatment of disease.

The Strominger and Wucherpfennig PCT application, and references citedtherein, all of which are incorporated by reference, describe the HLAclass II and TCR binding pockets which contact residues of an HLA classII peptide. By keeping the residues which are likely to bind in the HLAclass II and/or TCR binding pockets constant or permitting onlyspecified substitutions, functional variants of SSX HLA class II bindingpeptides can be prepared which retain binding to HLA class II and T cellreceptor.

Thus methods for identifying additional SSX family HLA class IIpeptides, in particular SSX-2 HLA class II binding peptides, andfunctional variants thereof, are provided. In general, any SSX proteincan be subjected to the analysis noted above, peptide sequences selectedand the tested as described herein. With respect to SSX-2, for example,the methods include selecting a SSX-2 HLA class II binding peptide, anHLA class II binding molecule which binds the SSX-2 HLA class II bindingpeptide, and a T cell which is stimulated by the SSX-2 HLA class IIbinding peptide presented by the HLA class II binding molecule. Inpreferred embodiments, the SSX-2 HLA class II binding peptide comprisesthe amino acid sequence of SEQ ID NOs:1-8. More preferably, the peptideconsists essentially of or consists of the amino acid sequences of SEQID NOs:1-7. The first amino acid residue of the SSX-2 HLA class IIbinding peptide is mutated to prepare a variant peptide. The amino acidresidue can be mutated according to the principles of HLA and T cellreceptor contact points set forth in the Strominger and WucherpfennigPCT application described above. Any method for preparing variantpeptides can be employed, such as synthesis of the variant peptide,recombinantly producing the variant peptide using a mutated nucleic acidmolecule, and the like.

The binding of the variant peptide to HLA class II binding molecules andstimulation of the T cell are then determined according to standardprocedures. For example, as exemplified below, the variant peptide canbe contacted with an antigen presenting cell which contains the HLAclass II molecule which binds the SSX-2 peptide to form a complex of thevariant peptide and antigen presenting cell. This complex can then becontacted with a T cell which recognizes the SSX-2 HLA class II bindingpeptide presented by the HLA class II binding molecule. T cells can beobtained from a patient having a condition characterized by expressionof SSX-2, such as cancer. Recognition of variant peptides by the T cellscan be determined by measuring an indicator of T cell stimulation suchas cytokine production (e.g., TNF or IFN-γ) or proliferation of the Tcells. Similar procedures can be carried out for identification andcharacterization of other SSX family HLA class II binding peptides.

Binding of a variant peptide to the HLA class II binding molecule andstimulation of the T cell by the variant peptide presented by the HLAclass II binding molecule indicates that the variant peptide is afunctional variant. The methods also can include the step of comparingthe stimulation of the T cell by the SSX-2 HLA class II binding peptideand the stimulation of the T cell by the functional variant as adetermination of the effectiveness of the stimulation of the T cell bythe functional variant. By comparing the functional variant with theSSX-2 HLA class II binding peptide, peptides with increased T cellstimulatory properties can be prepared.

The foregoing methods can be repeated sequentially with a second, third,fourth, fifth, sixth, seventh, eighth, ninth, and tenth substitutions toprepare additional functional variants of the disclosed SSX-2 HLA classII binding peptides.

Variants of the SSX-2 HLA class II binding peptides prepared by any ofthe foregoing methods can be sequenced, if necessary, to determine theamino acid sequence and thus deduce the nucleotide sequence whichencodes such variants.

Also a part of the invention are those nucleic acid sequences which codefor a SSX HLA class II binding peptides or variants thereof and othernucleic acid sequences which hybridize to a nucleic acid moleculeconsisting of the above described nucleotide sequences, under highstringency conditions. Preferred nucleic acid molecules include thosecomprising the nucleotide sequences that encode SEQ ID NOs:1-7, whichare SEQ ID NOs:15-21, respectively. The term “stringent conditions” asused herein refers to parameters with which the art is familiar Nucleicacid hybridization parameters may be found in references which compilesuch methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook,et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, New York, 1989, or Current Protocols in MolecularBiology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York.More specifically, high stringency conditions, as used herein, refers tohybridization at 65° C. in hybridization buffer (3.5×SSC, 0.02% Ficoll,0.02% Polyvinyl pyrolidone, 0.02% Bovine Serum Albumin, 25 mM NaH₂PO₄(pH7), 0.5% SDS, 2 mM EDTA). SSC is 0.15M Sodium Chloride/0.015M SodiumCitrate, pH 7; SDS is Sodium Dodecyl Sulphate; and EDTA is Ethylenediaminetetraacetic acid. After hybridization, the membrane upon whichthe DNA is transferred is washed at 2×SSC at room temperature and thenat 0.1-0.5×SSC/0.1×SDS at temperatures up to 68° C. Alternatively, highstringency to hybridization may be performed using a commerciallyavailable hybridization buffer, such as ExpressHyb™ buffer (Clontech)using hybridization and washing conditions described by themanufacturer.

There are other conditions, reagents, and so forth which can used, whichresult in a similar degree of stringency. The skilled artisan will befamiliar with such conditions, and thus they are not given here. It willbe understood, however, that the skilled artisan will be able tomanipulate the conditions in a manner to permit the clear identificationof homologs and alleles of nucleic acids encoding the SSX HLA class IIbinding peptides of the invention. The skilled artisan also is familiarwith the methodology for screening cells and libraries for expression ofsuch molecules which then are routinely isolated, followed by isolationof the pertinent nucleic acid molecule and sequencing.

In general homologs and alleles typically will share at least 75%nucleotide identity and/or at least 90% amino acid identity to thenucleic acids that encode a SSX-2 HLA class II binding peptide (such asSEQ ID NOs:l-7) or to the amino acid sequence of such a peptide,respectively. In some instances homologs and alleles will share at least90% nucleotide identity and/or at least 95% amino acid identity, inother embodiments homologs and alleles will share at least 95%nucleotide identity and/or at least 98% amino acid identity, in furtherembodiments homologs and alleles will share at least 97% nucleotideidentity and/or at least 99% amino acid identity and in still otherinstances will share at least 99% nucleotide identity and/or at least99.5% amino acid identity. Complements of the foregoing nucleic acidsalso are embraced by the invention.

In screening for nucleic acids which encode a SSX HLA class II bindingpeptide, a nucleic acid hybridization such as a Southern blot or aNorthern blot may be performed using the foregoing conditions, togetherwith a detectably labeled probe (e.g., radioactive such as ³²P,chemiluminescent, fluorescent labels). After washing the membrane towhich DNA encoding a SSX HLA class II binding peptide was finallytransferred, the membrane can be placed against X-ray film,phosphorimager or other detection device to detect the detectable label.

The invention also includes the use of nucleic acid sequences whichinclude alternative codons that encode the same amino acid residues ofthe SSX HLA class II binding peptides. For example, as disclosed herein,the peptide VGAQIPEKIQKAFDDIAKYFSK (SEQ ID NO:1) is a SSX-2 HLA class IIbinding peptide. The lysine residues (amino acids No. 8, 11, 18, and 22of SEQ ID NO:1) can be encoded by the codons AAA, and AAG. Each of thetwo codons is equivalent for the purposes of encoding a lysine residue.Thus, it will be apparent to one of ordinary skill in the art that anyof the lysine-encoding nucleotide triplets may be employed to direct theprotein synthesis apparatus, in vitro or in vivo, to incorporate alysine residue. Similarly, nucleotide sequence triplets which encodeother amino acid residues comprising the SSX-2 HLA class II bindingpeptide of SEQ ID NO:1 include: GUA, GUC, GUG and GUU (valine codons);GAA and GAG (glutamine codons); UUC and UUU (phenylalanine codons) andUAC and UAU (tyrosine codons). Other amino acid residues may be encodedsimilarly by multiple nucleotide sequences. Thus, the invention embracesdegenerate nucleic acids that differ from the native SSX HLA class IIbinding peptide encoding nucleic acids in codon sequence due to thedegeneracy of the genetic code.

It will also be understood that the invention embraces the use of thesequences in expression vectors, as well as to transfect host cells andcell lines, be these prokaryotic (e.g., E. coli), or eukaryotic (e.g.,dendritic cells, CHO cells, COS cells, yeast expression systems andrecombinant baculovirus expression in insect cells). The expressionvectors require that the pertinent sequence, i.e., those describedsupra, be operably linked to a promoter. As it has been found that humanHLA-DP molecules present a SSX-2 HLA class II binding peptide, theexpression vector may also include a nucleic acid sequence coding for anHLA-DP molecule. In a situation where the vector contains both codingsequences, it can be used to transfect a cell which does not normallyexpress either one. The SSX-2 HLA class II binding peptide codingsequence may be used alone, when, e.g. the host cell already expressesan HLA-DP molecule. Of course, there is no limit on the particular hostcell which can be used as the vectors which contain the two codingsequences may be used in host cells which do not express HLA-DPmolecules if desired, and the nucleic acid coding for the SSX-2 HLAclass II binding peptide can be used in antigen presenting cells whichexpress an HLA-DP molecule.

As used herein, “an HLA-DP molecule” includes the preferred subtypesDP101 and DP301 (i.e., DPB1*0101 AND DPB1*0301, including allelesDPB1*010101, DPB1*010102, DPB1*030101 and DPB1*030102), and othersubtypes known to one of ordinary skill in the art. Other subtypes,including those related to DP101 and DP301 can be found in various topublications and internet resources that update HLA allele lists.

As used herein, a “vector” may be any of a number of nucleic acids intowhich a desired sequence may be inserted by restriction and ligation fortransport between different genetic environments or for expression in ahost cell. Vectors are typically composed of DNA although RNA vectorsare also available. Vectors include, but are not limited to, plasmids,phagemids and virus genomes. A cloning vector is one which is able toreplicate autonomously or after integration into the genome in a hostcell, and which is further characterized by one or more endonucleaserestriction sites at which the vector may be cut in a determinablefashion and into which a desired DNA sequence may be ligated such thatthe new recombinant vector retains its ability to replicate in the hostcell. In the case of plasmids, replication of the desired sequence mayoccur many times as the plasmid increases in copy number within the hostbacterium or just a single time per host before the host reproduces bymitosis. In the case of phage, replication may occur actively during alytic phase or passively during a lysogenic phase. An expression vectoris one into which a desired DNA sequence may be inserted by restrictionand ligation such that it is operably joined to regulatory sequences andmay be expressed as an RNA transcript. Vectors may further contain oneor more marker sequences suitable for use in the identification of cellswhich have or have not been transformed or transfected with the vector.Markers include, for example, genes encoding proteins which increase ordecrease either resistance or sensitivity to antibiotics or othercompounds, genes which encode enzymes whose activities are detectable bystandard assays known in the art (e.g., β-galactosidase, luciferase oralkaline phosphatase), and genes which visibly affect the phenotype oftransformed or transfected cells, hosts, colonies or plaques (e.g.,green fluorescent protein). Preferred vectors are those capable ofautonomous replication and expression of the structural gene productspresent in the DNA segments to which they are operably joined.

Preferably the expression vectors contain sequences which target a SSXfamily polypeptide, e.g. SSX-2, or a HLA class II binding peptidederived therefrom, to the endosomes of a cell in which the protein orpeptide is expressed. HLA class II molecules contain an invariant chain(Ii) which impedes binding of other molecules to the HLA class IImolecules. This invariant chain is cleaved in endosomes, therebypermitting binding of peptides by HLA class II molecules. Therefore itis preferable that the SSX-2 HLA class II to binding peptides andprecursors thereof (e.g. the SSX-2 protein) are targeted to theendosome, thereby enhancing the binding of SSX-2 HLA class II bindingpeptide to HLA class II molecules. Targeting signals for directingmolecules to endosomes are known in the art and these signalsconveniently can be incorporated in expression vectors such that fusionproteins which contain the endosomal targeting signal are produced.Sanderson et al. (Proc. Nat'l. Acad. Sci. USA 92:7217-7221, 1995), Wu etal. (Proc. Nat'l. Acad. Sci. USA 92:11671-11675, 1995) and Thomson et al(J. Virol. 72:2246-2252, 1998) describe endosomal targeting signals(including invariant chain Ii and lysosomal-associated membrane proteinLAMP-1) and their use in directing antigens to endosomal and/orlysosomal cellular compartments.

Endosomal targeting signals such as invariant chain also can beconjugated to SSX-2 protein or peptides by non-peptide bonds (i.e. notfusion proteins) to prepare a conjugate capable of specificallytargeting SSX-2. Specific examples of covalent bonds include thosewherein bifunctional cross-linker molecules are used. The cross-linkermolecules may be homobifunctional or heterobifunctional, depending uponthe nature of the molecules to be conjugated. Homobifunctionalcross-linkers have two identical reactive groups. Heterobifunctionalcross-linkers are defined as having two different reactive groups thatallow for sequential conjugation reaction. Various types of commerciallyavailable cross-linkers are reactive with one or more of the followinggroups; primary amines, secondary amines, sulfhydryls, carboxyls,carbonyls and carbohydrates. One of ordinary skill in the art will beable to ascertain without undue experimentation the preferred moleculefor linking the endosomal targeting moiety and SSX-2 peptide or protein,based on the chemical properties of the molecules being linked and thepreferred characteristics of the bond or bonds.

As used herein, a coding sequence and regulatory sequences are said tobe “operably” joined when they are covalently linked in such a way as toplace the expression or transcription of the coding sequence under theinfluence or control of the regulatory sequences. If it is desired thatthe coding sequences be translated into a functional protein, two DNAsequences are said to be operably joined if induction of a promoter inthe 5′ regulatory sequences results in the transcription of the codingsequence and if the nature of the linkage between the two DNA sequencesdoes not (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the promoter region to direct the totranscription of the coding sequences, or (3) interfere with the abilityof the corresponding RNA transcript to be translated into a protein.Thus, a promoter region would be operably joined to a coding sequence ifthe promoter region were capable of effecting transcription of that DNAsequence such that the resulting transcript might be translated into thedesired protein or polypeptide.

The precise nature of the regulatory sequences needed for geneexpression may vary between species or cell types, but shall in generalinclude, as necessary, 5′ non-transcribed and 5′ non-translatedsequences involved with the initiation of transcription and translationrespectively, such as a TATA box, capping sequence, CAAT sequence, andthe like. In particular, such 5′ non-transcribed regulatory sequenceswill include a promoter region which includes a promoter sequence fortranscriptional control of the operably joined gene. Regulatorysequences may also include enhancer sequences or upstream activatorsequences as desired. The vectors of the invention may optionallyinclude 5′ leader or signal sequences. The choice and design of anappropriate vector is within the ability and discretion of one ofordinary skill in the art.

Expression vectors containing all the necessary elements for expressionare commercially available and known to those skilled in the art. See,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, 1989. Cells aregenetically engineered by the introduction into the cells ofheterologous DNA (RNA) encoding a SSX-2 HLA class II binding peptide.That heterologous DNA (RNA) is placed under operable control oftranscriptional elements to permit the expression of the heterologousDNA in the host cell. As described herein, such expression constructsoptionally also contain nucleotide sequences which encode endosomaltargeting signals, preferably human invariant chain or a targetingfragment thereof

Preferred systems for mRNA expression in mammalian cells are those suchas pRc/CMV and pcDNA3.1 (available from Invitrogen, Carlsbad, Calif.)that contain a selectable marker such as a gene that confers G418resistance (which facilitates the selection of stably transfected celllines) and the human cytomegalovirus (CMV) enhancer-promoter sequences.Additionally, suitable for expression in primate or canine cell lines isthe pCEP4 vector (Invitrogen), which contains an Epstein Barr virus(EBV) origin of replication, facilitating the maintenance of plasmid asa multicopy extrachromosomal element. Another expression vector is thepEF-BOS plasmid containing the promoter of polypeptide Elongation Factor1α, which stimulates efficiently transcription in vitro. The plasmid isdescribed by Mishizuma and Nagata (Nuc. Acids Res. 18:5322, 1990), andits use in transfection experiments is disclosed by, for example,Demoulin (Mol. Cell. Biol. 16:4710-4716, 1996). Still another preferredexpression vector is an adenovirus, described by Stratford-Perricaudet,which is defective for E1 and E3 proteins (J. Clin. Invest. 90:626-630,1992). The use of the adenovirus as an Adeno.P1A recombinant isdisclosed by Warnier et al., in intradermal injection in mice forimmunization against P1A (Int. J. Cancer, 67:303-310, 1996). Recombinantvectors including viruses selected from the group consisting ofadenoviruses, adeno-associated viruses, poxviruses including vacciniaviruses and attenuated poxviruses such as ALVAC, NYVAC, Semliki Forestvirus, Venezuelan equine encephalitis virus, retroviruses, Sindbisvirus, Ty virus-like particle, other alphaviruses, VSV, plasmids (e.g.“naked” DNA), bacteria (e.g. the bacterium Bacille Calmette Guerin,attenuated Salmonella), and the like can be used in such delivery, forexample, for use as a vaccine.

The invention also embraces so-called expression kits, which allow theartisan to prepare a desired expression vector or vectors. Suchexpression kits include at least separate portions of at least two ofthe previously discussed materials. Other components may be added, asdesired.

The invention as described herein has a number of uses, some of whichare described herein. The following uses are described for SSX-2 HLAclass II binding peptides but are equally applicable to use of other SSXfamily HLA class II binding peptides. First, the invention permits theartisan to diagnose a disorder characterized by expression of a SSX-2HLA class II binding peptide. These methods involve determiningexpression or presence in a biological sample of a SSX-2 HLA class IIbinding peptide, or a complex of a SSX-2 HLA class II binding peptideand an HLA class II molecule. The expression of a peptide or complex ofpeptide and HLA class II molecule can be determined by assaying with abinding partner for the peptide or complex, such as an antibody, a Tlymphocyte, a multimeric complex of T cell receptors specific for thecomplex, and the like. Assays that are well known in the immunologicalarts can be employed, such as ELISA, ELISPOT, flow cytometry, and thelike.

The invention further includes nucleic acid or protein microarrays withcomponents to that bind SSX-2 HLA class II peptides or nucleic acidsencoding such polypeptides. In this aspect of the invention, standardtechniques of microarray technology are utilized to assess expression ofthe SSX-2 polypeptides and/or identify biological constituents that bindsuch polypeptides. The constituents of biological samples includeantibodies, lymphocytes (particularly T lymphocytes), T cell receptormolecules and the like. Protein microarray technology, which is alsoknown by other names including: protein chip technology and solid-phaseprotein array technology, is well known to those of ordinary skill inthe art and is based on, but not limited to, obtaining an array ofidentified peptides or proteins on a fixed substrate, binding targetmolecules or biological constituents to the peptides, and evaluatingsuch binding. See, e.g., G. MacBeath and S. L. Schreiber, “PrintingProteins as Microarrays for High-Throughput Function Determination,”Science 289(5485):1760-1763, 2000. Nucleic acid arrays, particularlyarrays that bind SSX-2 peptides also can be used for diagnosticapplications, such as for identifying subjects that have a conditioncharacterized by SSX-2 polypeptide expression.

Microarray substrates include but are not limited to glass, silica,aluminosilicates, borosilicates, metal oxides such as alumina and nickeloxide, various clays, nitrocellulose, or nylon. The microarraysubstrates may be coated with a compound to enhance synthesis of a probe(peptide or nucleic acid) on the substrate. Coupling agents or groups onthe substrate can be used to covalently link the first nucleotide oramino acid to the substrate. A variety of coupling agents or groups areknown to those of skill in the art. Peptide or nucleic acid probes thuscan be synthesized directly on the substrate in a predetermined grid.Alternatively, peptide or nucleic acid probes can be spotted on thesubstrate, and in such cases the substrate may be coated with a compoundto enhance binding of the probe to the substrate. In these embodiments,presynthesized probes are applied to the substrate in a precise,predetermined volume and grid pattern, preferably utilizing acomputer-controlled robot to apply probe to the substrate in acontact-printing manner or in a non-contact manner such as ink jet orpiezo-electric delivery. Probes may be covalently linked to thesubstrate.

Targets are peptides or proteins and may be natural or synthetic. Thetissue may be obtained from a subject or may be grown in culture (e.g.from a cell line).

In some embodiments of the invention one or more control peptide orprotein molecules are attached to the substrate. Preferably, controlpeptide or protein molecules allow determination of factors such aspeptide or protein quality and binding characteristics, reagent qualityand effectiveness, binding success, and analysis thresholds and success.

Nucleic acid microarray technology, which is also known by other namesincluding: DNA chip technology, gene chip technology, and solid-phasenucleic acid array technology, is well known to those of ordinary skillin the art and is based on, but not limited to, obtaining an array ofidentified nucleic acid probes on a fixed substrate, labeling targetmolecules with reporter molecules (e.g., radioactive, chemiluminescent,or fluorescent tags such as fluorescein, Cye3-dUTP, or Cye5-dUTP),hybridizing target nucleic acids to the probes, and evaluatingtarget-probe hybridization. A probe with a nucleic acid sequence thatperfectly matches the target sequence will, in general, result indetection of a stronger reporter-molecule signal than will probes withless perfect matches. Many components and techniques utilized in nucleicacid microarray technology are presented in The Chipping Forecast,Nature Genetics, Vol. 21, January 1999, the entire contents of which isincorporated by reference herein.

According to the present invention, nucleic acid microarray substratesmay include but are not limited to glass, silica, aluminosilicates,borosilicates, metal oxides such as alumina and nickel oxide, variousclays, nitrocellulose, or nylon. In all embodiments a glass substrate ispreferred. According to the invention, probes are selected from thegroup of nucleic acids including, but not limited to: DNA, genomic DNA,cDNA, and oligonucleotides; and may be natural or synthetic.Oligonucleotide probes preferably are 20 to 25-mer oligonucleotides andDNA/cDNA probes preferably are 500 to 5000 bases in length, althoughother lengths may be used. Appropriate probe length may be determined byone of ordinary skill in the art by following art-known procedures. Inone embodiment, preferred probes are sets of two or more molecule thatbind the nucleic acid molecules that encode the SSX-2 HLA class IIbinding peptides set forth herein. Probes may be purified to removecontaminants using standard methods known to those of ordinary skill inthe art such as gel filtration or precipitation.

In one embodiment, the microarray substrate may be coated with acompound to enhance synthesis of the probe on the substrate. Suchcompounds include, but are not limited to, oligoethylene glycols. Inanother embodiment, coupling agents or groups on the substrate can beused to covalently link the first nucleotide or olignucleotide to thesubstrate. These to agents or groups may include, for example, amino,hydroxy, bromo, and carboxy groups. These reactive groups are preferablyattached to the substrate through a hydrocarbyl radical such as analkylene or phenylene divalent radical, one valence position occupied bythe chain bonding and the remaining attached to the reactive groups.These hydrocarbyl groups may contain up to about ten carbon atoms,preferably up to about six carbon atoms. Alkylene radicals are usuallypreferred containing two to four carbon atoms in the principal chain.These and additional details of the process are disclosed, for example,in U.S. Pat. No. 4,458,066, which is incorporated by reference in itsentirety.

In one embodiment, probes are synthesized directly on the substrate in apredetermined grid pattern using methods such as light-directed chemicalsynthesis, photochemical deprotection, or delivery of nucleotideprecursors to the substrate and subsequent probe production.

In another embodiment, the substrate may be coated with a compound toenhance binding of the probe to the substrate. Such compounds include,but are not limited to: polylysine, amino silanes, amino-reactivesilanes (Chipping Forecast, 1999) or chromium. In this embodiment,presynthesized probes are applied to the substrate in a precise,predetermined volume and grid pattern, utilizing a computer-controlledrobot to apply probe to the substrate in a contact-printing manner or ina non-contact manner such as ink jet or piezo-electric delivery. Probesmay be covalently linked to the substrate with methods that include, butare not limited to, UV-irradiation. In another embodiment probes arelinked to the substrate with heat.

Targets for microarrays are nucleic acids selected from the group,including but not limited to: DNA, genomic DNA, cDNA, RNA, mRNA and maybe natural or synthetic. In all embodiments, nucleic acid targetmolecules from human tissue are preferred. The tissue may be obtainedfrom a subject or may be grown in culture (e.g. from a cell line).

In embodiments of the invention one or more control nucleic acidmolecules are attached to the substrate. Preferably, control nucleicacid molecules allow determination of factors such as nucleic acidquality and binding characteristics, reagent quality and effectiveness,hybridization success, and analysis thresholds and success. Controlnucleic acids may include but are not limited to expression products ofgenes such as housekeeping genes or fragments thereof.

The invention also permits the artisan to treat a subject having adisorder characterized by expression of a SSX-2 HLA class II bindingpeptide. Treatments include administering an agent which enriches in thesubject a complex of a SSX-2 HLA class II binding peptide and an HLAclass II molecule, and administering CD4⁺ T lymphocytes which arespecific for such complexes. Agents useful in the foregoing treatmentsinclude SSX-2 HLA class II binding peptides and functional variantsthereof, proteins including such SSX-2 HLA class II binding peptides,optionally containing endosome targeting sequences fused to the SSX-2sequences, nucleic acids which express such proteins and peptides(including viruses and other vectors that contain the nucleic acids),complexes of such peptides and HLA class II binding molecules (e.g., HLADP), antigen presenting cells bearing complexes of a SSX-2 HLA class IIbinding peptide and an HLA class II binding molecule (such as dendriticcells bearing one or more SSX-2 HLA class II binding peptides bound toHLA class II molecules), and the like. The invention also permits anartisan to selectively enrich a population of T lymphocytes for CD4⁺ Tlymphocytes specific for a SSX-2 HLA class II binding peptide.

The isolation of the SSX-2 HLA class II binding peptides also makes itpossible to isolate and/or synthesize nucleic acids that encode theSSX-2 HLA class II binding peptides. Nucleic acids can be used toproduce in vitro or in prokaryotic or eukaryotic host cells the SSX-2HLA class II binding peptides.

Peptides comprising the SSX-2 HLA class II binding peptide of theinvention may be synthesized in vitro, using standard methods of peptidesynthesis, preferably automated peptide synthesis. In additiona, avariety of other methodologies well-known to the skilled practitionercan be utilized to obtain isolated SSX-2 HLA class II binding peptides.For example, an expression vector may be introduced into cells to causeproduction of the peptides. In another method, mRNA transcripts may bemicroinjected or otherwise introduced into cells to cause production ofthe encoded peptides. Translation of mRNA in cell-free extracts such asthe reticulocyte lysate system also may be used to produce peptides.Those skilled in the art also can readily follow known methods forisolating peptides in order to obtain isolated SSX-2 HLA class IIbinding peptides. These include, but are not limited to,immunochromatography, HPLC, size-exclusion chromatography, ion-exchangechromatography and immune-affinity chromatography.

These isolated SSX-2 HLA class II binding peptides, proteins whichinclude such peptides, or complexes of the peptides and HLA class IImolecules, such as HLA-DP, may be combined with materials such asadjuvants to produce vaccines useful in treating disorders characterizedby expression of the SSX-2 HLA class II binding peptide. Preferably,vaccines are prepared from antigen presenting cells that present theSSX-2 HLA class II binding peptide/HLA class II complexes on theirsurface, such as dendritic cells, B cells, non-proliferativetransfectants, etcetera. In all cases where cells are used as a vaccine,these can be cells transfected with coding sequences for one or both ofthe components necessary to stimulate CD4⁺ lymphocytes, or be cellswhich already express both molecules without the need for transfection.For example, autologous antigen presenting cells can be isolated from apatient and treated to obtain cells which present SSX-2 epitopes inassociation of HLA class I and HLA class II molecules. These cells wouldbe capable of stimulating both CD4⁺ and CD8⁺ cell responses. Suchantigen presenting cells can be obtained by infecting dendritic cellswith recombinant viruses encoding an Ii.SSX-2 fusion protein. Dendriticcells also can be loaded with HLA class I and HLA class II peptideepitopes.

Vaccines also encompass naked DNA or RNA, encoding a SSX-2 HLA class IIbinding peptide or precursor thereof, which may be produced in vitro andadministered via injection, particle bombardment, nasal aspiration andother methods. Vaccines of the “naked nucleic acid” type have beendemonstrated to provoke an immunological response including generationof CTLs specific for the peptide encoded by the naked nucleic acid(Science 259:1745-1748, 1993). Vaccines also include nucleic acidspackaged in a virus, liposome or other particle, including polymericparticles useful in drug delivery.

The immune response generated or enhanced by any of the treatmentsdescribed herein can be monitored by various methods known in the art.For example, the presence of T cells specific for a SSX-2 antigen can bedetected by direct labeling of T cell receptors with soluble fluorogenicMHC molecule tetramers which present the antigenic SSX-2 peptide (Altmanet al., Science 274:94-96, 1996; Dunbar et al., Curr. Biol. 8:413-416,1998). Briefly, soluble MHC class I molecules are folded in vitro in thepresence of β2-microglobulin and a peptide antigen which binds the classI molecule. After purification, the MHC/peptide complex is purified andlabeled with biotin. Tetramers are formed by mixing the biotinylatedpeptide-MHC complex with labeled avidin (e.g. phycoerythrin) at a molarratio of 4:1. to Tetramers are then contacted with a source of CTLs suchas peripheral blood or lymph node. The tetramers bind CTLs whichrecognize the peptide antigen/MHC class I complex. Cells bound by thetetramers can be sorted by fluorescence activated cell sorting toisolate the reactive CTLs. The isolated CTLs then can be expanded invitro for use as described herein. The use of MHC class II molecules astetramers was recently demonstrated by Crawford et al. (Immunity8:675-682, 1998; see also Dunbar and Ogg, J. Immunol. Methods268(1):3-7, 2002; Arnold et al., J. Immunol. Methods 271(1-2):137-151,2002). Multimeric soluble MHC class II molecules were complexed with acovalently attached peptide (which can be attached with or without alinker molecule), but peptides also can be loaded onto class IImolecules. The class II tetramers were shown to bind with appropriatespecificity and affinity to specific T cells. Thus tetramers can be usedto monitor both CD4⁺ and CD8⁺ cell responses to vaccination protocols.Methods for preparation of multimeric complexes of MHC class IImolecules are described in Hugues et al., J. Immunological Meth. 268:83-92, (2002) and references cited therein, each of which isincorporated by reference.

The SSX-2 HLA class II binding peptide, as well as complexes of SSX-2HLA class II binding peptide and HLA molecule, also may be used toproduce antibodies, using standard techniques well known to the art.Standard reference works setting forth the general principles ofantibody production include Catty, D., Antibodies, A Practical Approach,Vol. 1, IRL Press, Washington DC (1988); Klein, J., Immunology: TheScience of Cell-Non-Cell Discrimination, John Wiley and Sons, New York(1982); Kennett, R., et al., Monoclonal Antibodies, Hybridoma, A NewDimension In Biological Analyses, Plenum Press, New York (1980);Campbell, A., Monoclonal Antibody Technology, in Laboratory Techniquesand Biochemistry and Molecular Biology, Vol. 13 (Burdon, R. et al.EDS.), Elsevier Amsterdam (1984); and Eisen, H. N., Microbiology, thirdedition, Davis, B. D. et al. EDS. (Harper & Rowe, Philadelphia (1980).

Significantly, as is well-known in the art, only a small portion of anantibody molecule, the paratope, is involved in the binding of theantibody to its epitope (see, in general, Clark, W. R. (1986) TheExperimental Foundations of Modern Immunology Wiley & Sons, Inc., NewYork; Roitt, I. (1991) Essential Immunology, 7th Ed., BlackwellScientific Publications, Oxford). The pFc′ and Fc regions, for example,are effectors of the complement cascade but are not involved in antigenbinding. An antibody from which the pFc′ region has to beenenzymatically cleaved, or which has been produced without the pFc′region, designated an F(ab′)₂ fragment, retains both of the antigenbinding sites of an intact antibody. Similarly, an antibody from whichthe Fc region has been enzymatically cleaved, or which has been producedwithout the Fc region, designated an Fab fragment, retains one of theantigen binding sites of an intact antibody molecule. Proceedingfurther, Fab fragments consist of a covalently bound antibody lightchain and a portion of the antibody heavy chain denoted Fd. The Fdfragments are the major determinant of antibody specificity (a single Fdfragment may be associated with up to ten different light chains withoutaltering antibody specificity) and Fd fragments retain epitope-bindingability in isolation.

Within the antigen-binding portion of an antibody, as is well-known inthe art, there are complementarity determining regions (CDRs), whichdirectly interact with the epitope of the antigen, and framework regions(FRs), which maintain the tertiary structure of the paratope (see, ingeneral, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragmentand the light chain of IgG immunoglobulins, there are four frameworkregions (FR1 through FR4) separated respectively by threecomplementarity determining regions (CDR1 through CDR3). The CDRs, andin particular the CDR3 regions, and more particularly the heavy chainCDR3, are largely responsible for antibody specificity.

It is now well-established in the art that the non-CDR regions of amammalian antibody may be replaced with similar regions of nonspecificor heterospecific antibodies while retaining the epitopic specificity ofthe original antibody. This is most clearly manifested in thedevelopment and use of “humanized” antibodies in which non-human CDRsare covalently joined to human FR and/or Fc/pFc′ regions to produce afunctional antibody. See, e.g., U.S. Pat. Nos. 4,816,567, 5,225,539,5,585,089, 5,693,762 and 5,859,205.

Fully human monoclonal antibodies also can be prepared by immunizingmice transgenic for large portions of human immunoglobulin heavy andlight chain loci. See, e.g., U.S. Pat. Nos. 5,545,806, 6,150,584, andreferences cited therein. Following immunization of these mice (e.g.,XenoMouse (Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonalantibodies can be prepared according to standard hybridoma technology.These monoclonal antibodies will have human immunoglobulin amino acidsequences and therefore will not provoke human anti-mouse antibody(HAMA) responses when administered to humans.

Thus, as will be apparent to one of ordinary skill in the art, thepresent invention also provides for F(ab′)₂, Fab, Fv and Fd fragments;chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2and/or light chain CDR3 regions have been replaced by homologous humanor non-human sequences; chimeric F(ab′)₂ fragment antibodies in whichthe FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have beenreplaced by homologous human or non-human sequences; chimeric Fabfragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or lightchain CDR3 regions have been replaced by homologous human or non-humansequences; and chimeric Fd fragment antibodies in which the FR and/orCDR1 and/or CDR2 regions have been replaced by homologous human ornon-human sequences. The present invention also includes so-calledsingle chain antibodies.

The antibodies of the present invention thus are prepared by any of avariety of methods, including administering protein, fragments ofprotein, cells expressing the protein or fragments thereof and anappropriate HLA class II molecule, and the like to an animal to inducepolyclonal antibodies. The production of monoclonal antibodies isaccording to techniques well known in the art. Antibodies preparedaccording to the invention also preferably are specific for thepeptide/HLA complexes described herein.

The antibodies of this invention can be used for experimental purposes(e.g., localization of the HLA/peptide complexes, immunoprecipitations,Western blots, flow cytometry, ELISA etc.) as well as diagnostic ortherapeutic purposes (e.g., assaying extracts of tissue biopsies for thepresence of the SSX-2 peptides, HLA/peptide complexes, targetingdelivery of cytotoxic or cytostatic substances to cells expressing theappropriate HLA/peptide complex). The antibodies of this invention areuseful for the study and analysis of antigen presentation on tumor cellsand can be used to assay for changes in the HLA/peptide complexexpression before, during or after a treatment protocol, e.g.,vaccination with peptides, antigen presenting cells, HLA/peptidetetramers, adoptive transfer or chemotherapy.

The antibodies and antibody fragments of this invention may be coupledto diagnostic labeling agents for imaging of cells and tissues thatexpress the HLA/peptide complexes or may be coupled to therapeuticallyuseful agents by using standard methods well-known in the art. Theantibodies also may be coupled to labeling agents for imaging e.g.,radiolabels or fluorescent labels, or may be coupled to, e.g., biotin orantitumor agents, e.g., radioiodinated compounds, toxins such as ricin,methotrexate, cytostatic or cytolytic drugs, etc. Examples of diagnosticagents suitable for conjugating to the antibodies of this inventioninclude e.g., to barium sulfate, diatrizoate sodium, diatrizoatemeglumine, iocetamic acid, iopanoic acid, ipodate calcium, metrizamide,tyropanoate sodium and radiodiagnostics including positron emitters suchas fluorine-18 and carbon-11, gamma emitters such as iodine-123,technitium-99m, iodine-131 and indium-111, nuclides for nuclear magneticresonance such as fluorine and gadolinium. As used herein,“therapeutically useful agents” include any therapeutic molecules, whichare preferably targeted selectively to a cell expressing the HLA/peptidecomplexes, including antineoplastic agents, radioiodinated compounds,toxins, other cytostatic or cytolytic drugs. Antineoplastic therapeuticsare well known and include: aminoglutethimide, azathioprine, bleomycinsulfate, busulfan, carmustine, chlorambucil, cisplatin,cyclophosphamide, cyclosporine, cytarabidine, dacarbazine, dactinomycin,daunorubicin, doxorubicin, taxol, etoposide, fluorouracil,interferon-.alpha., lomustine, mercaptopurine, methotrexate, mitotane,procarbazine HCl, thioguanine, vinblastine sulfate and vincristinesulfate. Additional antineoplastic agents include those disclosed inChapter 52, Antineoplastic Agents (Paul Calabresi and Bruce A. Chabner),and the introduction thereto, 1202-1263, of Goodman and Gilman's “ThePharmacological Basis of Therapeutics”, Eighth Edition, 1990,McGraw-Hill, Inc. (Health Professions Division). Toxins can be proteinssuch as, for example, pokeweed anti-viral protein, cholera toxin,pertussis toxin, ricin, gelonin, abrin, diphtheria exotoxin, orPseudomonas exotoxin. Toxin moieties can also be high energy-emittingradionuclides such as cobalt-60. The antibodies may be administered to asubject having a pathological condition characterized by thepresentation of the HLA/peptide complexes of this invention, e.g.,melanoma or other cancers, in an amount sufficient to alleviate thesymptoms associated with the pathological condition.

When “disorder” or “condition” is used herein, it refers to anypathological condition where the SSX-2 HLA class II binding peptide isexpressed. Such disorders include cancers, such as biliary tract cancer;bladder cancer; breast cancer; brain cancer including glioblastomas andmedulloblastomas; cervical cancer; choriocarcinoma; colon cancerincluding colorectal carcinomas; endometrial cancer; esophageal cancer;gastric cancer; head and neck cancer; hematological neoplasms includingacute lymphocytic and myelogenous leukemia, multiple myeloma,AIDS-associated leukemias and adult T-cell leukemia lymphoma;intraepithelial neoplasms including Bowen's disease and Paget's disease;liver cancer; lung cancer including small cell lung cancer and non-smallcell lung cancer; to lymphomas including Hodgkin's disease andlymphocytic lymphomas; neuroblastomas; oral cancer including squamouscell carcinoma; osteosarcomas; ovarian cancer including those arisingfrom epithelial cells, stromal cells, germ cells and mesenchymal cells;pancreatic cancer; prostate cancer; rectal cancer; sarcomas includingleiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, synovialsarcoma and osteosarcoma; skin cancer including melanomas, Kaposi'ssarcoma, basocellular cancer, and squamous cell cancer; testicularcancer including germinal tumors such as seminoma, non-seminoma(teratomas, choriocarcinomas), stromal tumors, and germ cell tumors;thyroid cancer including thyroid adenocarcinoma and medullar carcinoma;transitional cancer and renal cancer including adenocarcinoma and Wilmstumor.

Some therapeutic approaches based upon the disclosure are premised oninducing a response by a subject's immune system to SSX HLA class IIbinding peptide presenting cells. One such approach is theadministration of autologous CD4⁺ T cells specific to the complex ofSSX-2 HLA class II binding peptide and an HLA class II molecule to asubject with abnormal cells of the phenotype at issue. It is within theskill of the artisan to develop such CD4⁺ T cells in vitro. Generally, asample of cells taken from a subject, such as blood cells, are contactedwith a cell presenting the complex and capable of provoking CD4⁺ Tlymphocytes to proliferate. The target cell can be a transfectant, suchas a COS cell, or an antigen presenting cell bearing HLA class IImolecules, such as dendritic cells or B cells. These transfectantspresent the desired complex of their surface and, when combined with aCD4⁺ T lymphocyte of interest, stimulate its proliferation. COS cellsare widely available, as are other suitable host cells. Specificproduction of CD4⁺ T lymphocytes is described below. The clonallyexpanded autologous CD4⁺ T lymphocytes then are administered to thesubject. The CD4⁺ T lymphocytes then stimulate the subject's immuneresponse, thereby achieving the desired therapeutic goal.

CTL proliferation can be increased by increasing the level of tryptophanin T cell cultures, by inhibiting enzymes which catabolizes tryptophan,such as indoleamine 2,3-dioxygenase (MO), or by adding tryptophan to theculture (see, e.g., PCT application WO99/29310). Proliferation of Tcells is enhanced by increasing the rate of proliferation and/orextending the number of divisions of the T cells in culture. Inaddition, increasing tryptophan in T cell cultures also enhances thelytic activity of the T cells grown in culture.

The foregoing therapy assumes that at least some of the subject'sabnormal cells present the relevant HLA/peptide complex. This can bedetermined very easily, as the art is very familiar with methods foridentifying cells which present a particular HLA molecule, as well ashow to identify cells expressing DNA of the pertinent sequences, in thiscase a SSX-2 sequence.

The foregoing therapy is not the only form of therapy that is availablein accordance with the invention. CD4⁺ T lymphocytes can also beprovoked in vivo, using a number of approaches. One approach is the useof non-proliferative cells expressing the complex. The cells used inthis approach may be those that normally express the complex, such asdendritic cells or cells transfected with one or both of the genesnecessary for presentation of the complex. Chen et al., (Proc. Natl.Acad. Sci. USA 88: 110-114, 1991) exemplifies this approach, showing theuse of transfected cells expressing HPV-E7 peptides in a therapeuticregime. Various cell types may be used. Similarly, vectors carrying oneor both of the genes of interest may be used. Viral or bacterial vectorsare especially preferred. For example, nucleic acids which encode aSSX-2 HLA class II binding peptide may be operably linked to promoterand enhancer sequences which direct expression of the SSX-2 HLA class IIbinding peptide in certain tissues or cell types. The nucleic acid maybe incorporated into an expression vector. Expression vectors may beunmodified extrachromosomal nucleic acids, plasmids or viral genomesconstructed or modified to enable insertion of exogenous nucleic acids,such as those encoding SSX-2 HLA class II binding peptides. Nucleicacids encoding a SSX-2 HLA class II binding peptide also may be insertedinto a retroviral genome, thereby facilitating integration of thenucleic acid into the genome of the target tissue or cell type. In thesesystems, the gene of interest is carried by a microorganism, e.g., aVaccinia virus, poxviruses in general, adenovirus, herpes simplex virus,retrovirus or the bacteria BCG, and the materials de facto “infect” hostcells. The cells which result present the complex of interest, and arerecognized by autologous CD4⁺ T cells, which then proliferate.

A similar effect can be achieved by combining a SSX HLA class II bindingpeptide with an adjuvant to facilitate incorporation into HLA class IIpresenting cells in vivo. If larger than the HLA class II bindingportion (e.g., SEQ ID NOs:1-7), the SSX-2 HLA class II binding peptidecan be processed if necessary to yield the peptide partner of the HLAmolecule while the peptides disclosed herein are believed to bepresented without the need for further processing. Generally, subjectscan receive an intradermal injection of an effective amount of the SSX-2HLA class II binding peptide. Initial doses can be followed by boosterdoses, following immunization protocols standard in the art.

A preferred method for facilitating incorporation of SSX-2 HLA class IIbinding peptides into HLA class II presenting cells is by expressing inthe presenting cells a polypeptide which includes an endosomal targetingsignal fused to a SSX-2 polypeptide which includes the class II bindingpeptide. Particularly preferred are SSX-2 fusion proteins which containhuman invariant chain Ii.

Any of the foregoing compositions or protocols can include also SSX HLAclass I binding peptides for induction of a cytolytic T lymphocyteresponse. For example, the SSX-2 protein can be processed in a cell toproduce both HLA class I and HLA class II responses. SSX-2 peptides havebeen described in U.S. Pat. No. 6,548,064, and by Ayyoub et al. (JImmunol 168(4):1717-22, 2002) and Rubio-Godoy et al. (Eur J Immunol.32:2292-2299, 2002). SSX gene and protein family members are disclosedin U.S. Pat. Nos. 6,291,658 and 6,339,140. By administering SSX-2peptides which bind HLA class I and class II molecules (or nucleic acidencoding such peptides), an improved immune response may be provided byinducing both T helper cells and cytotoxic T cells.

In addition, non-SSX-2 tumor associated peptides also can beadministered to increase immune response via HLA class I and/or classII. It is well established that cancer cells can express more that onetumor associated gene. It is within the scope of routine experimentationfor one of ordinary skill in the art to determine whether a particularsubject expresses additional tumor associated genes, and then includeHLA class I and/or HLA class II binding peptides derived from expressionproducts of such genes in the foregoing SSX-2 compositions and vaccines.

Especially preferred are nucleic acids encoding a series of epitopes,known as “polytopes”. The epitopes can be arranged in sequential oroverlapping fashion (see, e.g., Thomson et al., Proc. Natl. Acad. Sci.USA 92:5845-5849, 1995; Gilbert et al., Nature Biotechnol. 15:1280-1284,1997), with or without the natural flanking sequences, and can beseparated by unrelated linker sequences if desired. The polytope isprocessed to generated individual epitopes which are recognized by theimmune system for generation of immune responses.

Thus, for example, SSX-2 HLA class II binding peptides can be combinedwith peptides from other tumor rejection antigens (e.g. by preparationof hybrid nucleic acids or polypeptides) and with SSX-2 HLA class Ibinding peptides (some of which are listed below) to form “polytopes”.Exemplary tumor associated peptide antigens that can be administered toinduce or enhance an immune response are derived from tumor associatedgenes and encoded proteins including MAGE-A1, MAGE-A2, MAGE-2, MAGE-A4,MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11,MAGE-A12, MAGE-A13, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6,GAGE-7, GAGE-8, BAGE-1, RAGE-1, LB33/MUM-1, PRAME, NAG, MAGE-Xp2(MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), tyrosinase, brainglycogen phosphorylase, Melan-A, MAGE-C1, MAGE-C2, NY-ESO-1, LAGE-1,SSX-1, SSX-3, SSX-4, SSX-5, SCP-1 and CT-7. For example, antigenicpeptides characteristic of tumors include those listed in published PCTapplication WO 00/20581 (PCT/US99/21230).

Other examples of HLA class I and HLA class II binding peptides will beknown to one of ordinary skill in the art (for example, see Coulie, StemCells 13:393-403, 1995), and can be used in the invention in a likemanner as those disclosed herein. One of ordinary skill in the art canprepare polypeptides comprising one or more SSX-2 peptides and one ormore of the foregoing tumor rejection peptides, or nucleic acidsencoding such polypeptides, according to standard procedures ofmolecular biology.

Thus polytopes are groups of two or more potentially immunogenic orimmune response stimulating peptides which can be joined together invarious arrangements (e.g. concatenated, overlapping). The polytope (ornucleic acid encoding the polytope) can be administered in a standardimmunization protocol, e.g. to animals, to test the effectiveness of thepolytope in stimulating, enhancing and/or provoking an immune response.

The peptides can be joined together directly or via the use of flankingsequences to form polytopes, and the use of polytopes as vaccines iswell known in the art (see, e.g., Thomson et al., Proc. Acad. Natl.Acad. Sci USA 92(13):5845-5849, 1995; Gilbert et al., Nature Biotechnol.15(12):1280-1284, 1997; Thomson et al., J. Immunol. 157(2):822-826,1996; Tam et al., J. Exp. Med. 171(1):299-306, 1990). For example, Tamshowed that polytopes consisting of both MHC class I and class IIbinding epitopes successfully generated antibody and protective immunityin a mouse model. Tam also demonstrated that polytopes comprising“strings” of epitopes are processed to yield individual epitopes whichare presented by MHC molecules and recognized by CTLs. Thus polytopescontaining various numbers and combinations of epitopes can be preparedand tested for recognition by CTLs and for efficacy in increasing animmune response.

It is known that tumors express a set of tumor antigens, of which onlycertain subsets may be expressed in the tumor of any given patient.Polytopes can be prepared which correspond to the different combinationof epitopes representing the subset of tumor rejection antigensexpressed in a particular patient. Polytopes also can be prepared toreflect a broader spectrum of tumor rejection antigens known to beexpressed by a tumor type. Polytopes can be introduced to a patient inneed of such treatment as polypeptide structures, or via the use ofnucleic acid delivery systems known in the art (see, e.g., Allsopp etal., Eur. J. Immunol. 26(8):1951-1959, 1996). Adenovirus, pox virus,Ty-virus like particles, adeno-associated virus, plasmids, bacteria,etc. can be used in such delivery. One can test the polytope deliverysystems in mouse models to determine efficacy of the delivery system.The systems also can be tested in human clinical trials.

As part of the immunization compositions, one or more substances thatpotentiate an immune response are administered along with the peptidesdescribed herein. Such substances include adjuvants and cytokines. Anadjuvant is a substance incorporated into or administered with antigenwhich potentiates the immune response. Adjuvants may enhance theimmunological response by providing a reservoir of antigen(extracellularly or within macrophages), activating macrophages andstimulating specific sets of lymphocytes. Adjuvants of many kinds arewell known in the art. Specific examples of adjuvants includemonophosphoryl lipid A (MPL, SmithKline Beecham), a congener obtainedafter purification and acid hydrolysis of Salmonella minnesota Re 595lipopolysaccharide; saponins including QS21 (SmithKline Beecham), a pureQA-21 saponin purified from Quillja saponaria extract; DQS21, describedin PCT application WO96/33739 (SmithKline Beecham); QS-7, QS-17, QS-18,and QS-L1 (So et al., Mol. Cells 7:178-186, 1997); incomplete Freund'sadjuvant; complete Freund's adjuvant; montanide; immunostimulatoryoligonucleotides (see e.g. CpG oligonucleotides described by Kreig etal., Nature 374:546-9, 1995); reagents that bind to one of the toll-likereceptors; vitamin E and various water-in-oil emulsions prepared frombiodegradable oils such as squalene and/or tocopherol. Preferably, thepeptides are to administered mixed with a combination of DQS21/MPL. Theratio of DQS21 to MPL typically will be about 1:10 to 10:1, preferablyabout 1:5 to 5:1 and more preferably about 1:1. Typically for humanadministration, DQS21 and MPL will be present in a vaccine formulationin the range of about 1 μg to about 100 μg. Other adjuvants are known inthe art and can be used in the invention (see, e.g. Goding, MonoclonalAntibodies: Principles and Practice, 2nd Ed., 1986). Methods for thepreparation of mixtures or emulsions of peptide and adjuvant are wellknown to those of skill in the art of vaccination.

Other agents which stimulate the immune response of the subject can alsobe administered to the subject. For example, other cytokines are alsouseful in vaccination protocols as a result of their lymphocyteregulatory properties. Many other cytokines useful for such purposeswill be known to one of ordinary skill in the art, includinginterleukin-12 (IL-12) which has been shown to enhance the protectiveeffects of vaccines (see, e.g., Science 268: 1432-1434, 1995), GM-CSFand IL-18. Thus cytokines can be administered in conjunction withantigens and adjuvants to increase the immune response to the antigens.There are a number of additional immune response potentiating compoundsthat can be used in vaccination protocols. These include costimulatorymolecules provided in either protein or nucleic acid form. Suchcostimulatory molecules include the B7-1 and B7-2 (CD80 and CD86respectively) molecules which are expressed on dendritic cells (DC) andinteract with the CD28 molecule expressed on the T cell. Thisinteraction provides costimulation (signal 2) to an antigen/MHC/TCRstimulated (signal 1) T cell, increasing T cell proliferation, andeffector function. B7 also interacts with CTLA4 (CD152) on T cells andstudies involving CTLA4 and B7 ligands indicate that the B7-CTLA4interaction can enhance antitumor immunity and CTL proliferation (Zhenget al., Proc. Nat'l Acad. Sci. USA 95:6284-6289, 1998).

B7 typically is not expressed on tumor cells so they are not efficientantigen presenting cells (APCs) for T cells. Induction of B7 expressionwould enable the tumor cells to stimulate more efficiently CTLproliferation and effector function. A combination of B7/IL-6/IL-12costimulation has been shown to induce IFN-gamma and a Th1 cytokineprofile in the T cell population leading to further enhanced T cellactivity (Gajewski et al., J. Immunol. 154:5637-5648, 1995). Tumor celltransfection with B7 has been discussed in relation to in vitro CTLexpansion for adoptive transfer immunotherapy by Wang et al. (J.Immunother. 19:1-8, 1996). Other delivery mechanisms for the B7 moleculewould include nucleic acid (naked DNA) immunization (Kim et al., NatureBiotechnol. 15:7:641-646, 1997) and recombinant viruses such as adenoand pox (Wendtner et al., Gene Ther. 4:726-735, 1997). These systems areall amenable to the construction and use of expression cassettes for thecoexpression of B7 with other molecules of choice such as the antigensor fragment(s) of antigens discussed herein (including polytopes) orcytokines. These delivery systems can be used for induction of theappropriate molecules in vitro and for in vivo vaccination situations.The use of anti-CD28 antibodies to directly stimulate T cells in vitroand in vivo could also be considered. Similarly, the inducibleco-stimulatory molecule ICOS which induces T cell responses to foreignantigen could be modulated, for example, by use of anti-ICOS antibodies(Hutloff et al., Nature 397:263-266, 1999).

Lymphocyte function associated antigen-3 (LFA-3) is expressed on APCsand some tumor cells and interacts with CD2 expressed on T cells. Thisinteraction induces T cell IL-2 and IFN-gamma production and can thuscomplement but not substitute, the B7/CD28 costimulatory interaction(Parra et al., J. Immunol., 158:637-642, 1997; Fenton et al., J.Immunother., 21:95-108, 1998).

Lymphocyte function associated antigen-1 (LFA-1) is expressed onleukocytes and interacts with ICAM-1 expressed on APCs and some tumorcells. This interaction induces T cell IL-2 and IFN-gamma production andcan thus complement but not substitute, the B7/CD28 costimulatoryinteraction (Fenton et al., 1998). LFA-1 is thus a further example of acostimulatory molecule that could be provided in a vaccination protocolin the various ways discussed above for B7.

Complete CTL activation and effector function requires Th cell helpthrough the interaction between the Th cell CD40L (CD40 ligand) moleculeand the CD40 molecule expressed by DCs (Ridge et al., Nature 393:474,1998; Bennett et al., Nature 393:478, 1998; Schoenberger et al., Nature393:480, 1998). This mechanism of this costimulatory signal is likely toinvolve upregulation of B7 and associated IL-6/IL-12 production by theDC (APC). The CD40-CD40L interaction thus complements the signal 1(antigen/MHC-TCR) and signal 2 (B7-CD28) interactions.

The use of anti-CD40 antibodies to stimulate DC cells directly, would beexpected to enhance a response to tumor associated antigens which arenormally encountered outside of an inflammatory context or are presentedby non-professional APCs (tumor cells). Other methods for inducingmaturation of dendritic cells, e.g., by increasing CD40-CD40Linteraction, or by contacting DCs with CpG-containingoligodeoxynucleotides or stimulatory sugar moieties from extracellularmatrix, are known in the art. In these situations Th help and B7costimulation signals are not provided. This mechanism might be used inthe context of antigen pulsed DC based therapies or in situations whereTh epitopes have not been defined within known tumor associated antigenprecursors.

When administered, the therapeutic compositions of the present inventionare administered in pharmaceutically acceptable preparations. Suchpreparations may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, supplementary immune potentiating agents such as adjuvants andcytokines and optionally other therapeutic agents.

The preparations of the invention are administered in effective amounts.An effective amount is that amount of a pharmaceutical preparation thatalone, or together with further doses, stimulates the desired response.In the case of treating cancer, the desired response is inhibiting theprogression of the cancer. This may involve only slowing the progressionof the disease temporarily, although more preferably, it involveshalting the progression of the disease permanently. In the case ofinducing an immune response, the desired response is an increase inantibodies or T lymphocytes which are specific for the SSX-2immunogen(s) employed. These desired responses can be monitored byroutine methods or can be monitored according to diagnostic methods ofthe invention discussed herein.

Where it is desired to stimulate an immune response using a therapeuticcomposition of the invention, this may involve the stimulation of ahumoral antibody response resulting in an increase in antibody titer inserum, a clonal expansion of cytotoxic lymphocytes, or some otherdesirable immunologic response. It is believed that doses of immunogensranging from one nanogram/kilogram to 100 milligrams/kilogram, dependingupon the mode of administration, would be effective. The preferred rangeis believed to be between 500 nanograms and 500 micrograms per kilogram.The absolute amount will depend upon a variety of factors, including thematerial selected for administration, whether the administration is insingle or multiple doses, and individual patient parameters includingage, physical condition, size, weight, and the stage of the disease.These factors are well known to to those of ordinary skill in the artand can be addressed with no more than routine experimentation.

EXAMPLES

Because of its expression in different tumor types, the cancer/testisantigen SSX-2 is among the most promising candidates for the developmentof generic cancer vaccines. SSX-2 is a classical cancer testis antigenbelonging to a multigene family mapping to chromosome X. Some familymembers, including SSX-2, are expressed in a wide variety of tumors(Naka et al. Int J Cancer 2002. 98:640-642). The SSX-2 encoding gene wasinitially described as one of two partner genes found in a recurrentchromosomal translocation in synovial sarcoma (Clark et al. Nat Genet.1994. 7:502-508; Crew et al. EMBO J. 1995. 14:2333-2340), and morerecently identified by SEREX analysis of serum from a melanoma patient.The potential spontaneous immunogenicity of the SSX-2 antigen wasinitially suggested by detection of specific antibodies in 10% ofmelanoma patients (Tureci et al, Cancer Res 1996. 56:4766-4772). Byanalyzing CD8⁺ T lymphocytes from an SSX-2 expressing melanoma patient,an epitope was identified mapping to the 41-49 region of the SSX-2protein and recognized by tumor-reactive CD8⁺ T lymphocytes inassociation with the MHC Class I allele HLA-A2 (Rubio-Godoy et al., EurJ Immunol. 32:2292-2299, 2002; Ayyoub et al. J Immunol 168:1717-1722,2002) Importantly, whereas a large functional avidity of antigenrecognition and tumor reactivity has been found among isolated SSX241-49 specific CD8⁺ T cells, those isolated from both tumor infiltratingand circulating lymphocytes of patients bearing SSX-2 expressing tumorlesions uniformly exhibited high functional avidity of antigenrecognition and tumor reactivity. These findings indicate thatspontaneous T cell responses to SSX-2 frequently occur in antigenexpressing melanoma patients, encouraging the search for additional MHCclass I and class II restricted epitopes in this patient population.

The first CD4⁺ T cell epitope encoded by SSX-2 now has been identified,as described herein. The identified epitope mapped to the 19-34 regionof the protein and was recognized by CD4⁺ T cells from an antigenexpressing melanoma patient in association with HLA-DP. The absence ofdetectable response in healthy donors and other patients suggests thatSSX-2 specific CD4⁺ T cells in the responder patient had been previouslyexpanded in vivo in response to the autologous tumor. Interestingly, theepitope did not appear to be presented on the surface of tumor cells atlevels sufficient to allow direct recognition. In contrast, it wasefficiently presented by autologous dendritic cells, supporting theconcept that processing by professional APC is the main pathway throughwhich CD4⁺ T cell immunoresponse to tumors antigens occurs in vivo.

Materials and Methods

Cell lines and tissue culture. Melanoma cell lines and anti HLA-DR(D1.12), HLA-DP (B7.21.3) and HLA-DQ (BT3/4) antibodies were kindlyprovided by Dr. D. Rimoldi (Ludwig Institute for Cancer Research,Lausanne, Switzerland). Cell lines were maintained in RPMI 1640 (LifeTechnologies, Gaithersburg, MD) supplemented with 10% heat inactivatedfetal calf serum (FCS). Culture medium for lymphocytes was IMDM (LifeTechnologies, Basel, Switzerland) supplemented with 8% heat inactivatedpooled human serum (CTL medium), human recombinant IL-2 (Glaxo, Geneva,Switzerland) and IL-7 (Biosource International, Camarillo, Calif.).

Generation of SSX-2 specific CD4⁺ T cells. In vitro sensitization ofCD4⁺ T cells was carried out as described previously for CD8⁺ T cells.Briefly, 1 to 2×10⁶ CD4⁺ T cells highly enriched from peripheral bloodmononuclear cells (PBMC) by magnetic cell sorting using a miniMACSdevice (Miltenyi Biotec, Sunnyvale, Calif., USA) were stimulated with amixture containing peptides spanning the entire SSX-2 protein sequence(Ayyoub et al, J Immunol 2002. 32:2292-2299) (2 μM each) in the presenceof irradiated autologous cells from the CD4⁻ fraction in CTL mediumcontaining rhIL-2 (10 U/ml) and IL-7 (10 pg/ml). The enriched CD4⁺ Tcells were cultured 2-3 weeks prior to being tested. CD4⁺ T cellssecreting cytokines in response to peptide stimulation were isolated bycytokine guided flow cytometry cell sorting using the cytokine secretiondetection kit (Miltenyi Biotec) and cloned by limiting dilution culturein the presence of PHA (Sigma), allogenic irradiated PBMC and rhIL-2 asdescribed (Valmori et al., 2001, Cancer Res. 61:501-512). Clones weresubsequently expanded by periodic (3-4 weeks) stimulation under the sameconditions.

Molecular typing of HLA-DP molecules, construction of plasmids andtransient transfection. Primers pairs used for the PCR amplification ofHLA-DP alleles were as follows: DPA forward primer: ATG CGC CCT GAA GACAGA ATG T (SEQ ID NO:22); to DPA reverse primer: TCA CAG GGT CCC CTG GGCCCG GGG GA (SEQ ID NO:23); DPB forward primer: ATG ATG GTT CTG CAG GTTTCT G (SEQ ID NO:24); and DPB reverse primer: TTA TGC AGA TCC TCG TTGAAC TTT C (SEQ ID NO:25). Obtained sequences were searched against theIMGT-HLA database to confirm the HLA-DP identity (hosted at the EuropeanBioinformatics Institute (EMBL-EBI) website). The SSX-2 plasmidcontained the SSX-2 cDNA cloned into pcDNA3.1 vector. Tumor cells weretransiently transfected with plasmids using FuGENE according to themanufacture's instructions (Roche Diagnostics, Rotkreuz, Switzerland).

Antigen recognition assays. For intracellular cytokine secretiondetection T cells were incubated with APC at a 1:1 T cells:APC ratioduring 4-6 h in the absence or in the presence of peptides at theindicated dose. One hour after the beginning of the incubation BrefeldinA (20 μg/ml, Sigma Chemical Co., Steinheim, Germany) was added toinhibit cytokine secretion. At the end of the incubation period cellswere stained with anti-CD4 mAb for 20 min at 4° C. and fixed. Cells werethen permeabilized using saponine (Sigma, 0.1% in PBS 10% FCS) stainedby incubation with mAb against IFN-γ or IL-2 (BD, Pharmingen) andanalyzed by flow cytometry. Data analysis was performed using Cell Questsoftware. For detection of cytokine secretion in the culture supernatantT cells (10,000) were incubated with stimulating cells (15,000/well) in96-well round-bottom plates in 200 μl/well of IMDM containing 10% humanserum and 20 U/ml hrIL2. After 24 h incubation at 37° C., culturesupernatants were collected and the content of IFN-γ determined by ELISA(BioSource Europe, Fleurus, Belgium). IFN-γ ELISPOT assay was performedas described previously (Ayyoub M. et al, 2002, J Immunol 32:2292-2299)using nitrocellulose-lined 96-well microplates (MAHA S45: Millipore,Bedford, Mass.) and an IFN-γ ELISPOT kit (DIACLONE, Besancon, France).Stimulator cells (5×10⁴/well) were added together with the indicatednumber of T cells and peptide (2 μM) where indicated. Spots were countedusing a stereomicroscope with a magnification of ×15.

Results Example 1 Assessment of SSX-2 Specific CD4⁺ T Cell Responses inCirculating Lymphocytes of Antigen Expressing Melanoma Patients

Enriched CD4⁺ T cells from PBMC samples from 5 melanoma patients withdetectable SSX-2 expression in their tumor lesions were stimulated invitro with a peptide mix containing 15 20-22 amino acid long peptidesspanning the SSX-2 protein sequence and overlapping by 10 amino acids(Ayyoub M. et al, 2002, J Immunol 32:2292-2299). Two to 3 weeks after asingle in vitro stimulation, culture aliquots were stimulated withsubmixtures, each composed of 3 peptides; P1-3 was composed of thepeptides as set forth in SEQ ID NO:26, SEQ ID NO:1, SEQ ID NO:27; P4-6was composed of the peptides as set forth in SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30; P7-9 was composed of the peptides as set forth in SEQ IDNO:31, SEQ ID NO:32, SEQ ID NO:33; P10-12 was composed of the peptidesas set forth in SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36; and P13-15 wascomposed of the peptides as set forth in SEQ ID NO:37, SEQ ID NO:38, SEQID NO:39. The presence of specific CD4⁺ T cells was monitored byintracellular staining with cytokine specific antibodies (IFN-γ, IL-2,FIG. 1 and Table I). For one patient (LAU 672), one peptide submixture(P1-3, containing peptides SEQ ID NO:26, SEQ ID NO:1 and SEQ ID NO:27)stimulated a significant proportion of IFN-γ and IL-2 secreting CD4⁺ Tcells, as compared to controls containing either no peptide or otherpeptide mixtures (FIG. 1 and Table I). No specific responses weredetected in the case of the other 4 melanoma patients analyzed (notshown). Assessment of reactivity of the culture from patient LAU 672 tosingle peptides in the submixture P1-3 (SEQ ID NO:26, SEQ ID NO:1, SEQID NO:27) revealed that SEQ ID NO:1 was the active peptide, whereas nosignificant activity was detected in response to peptides SEQ ID NO:22and SEQ ID NO:23 (not shown). Peptide SSX-2 13-34 (SEQ ID NO:1) specificCD4⁺ T cells were isolated from the culture by cytokine secretion guidedflow cytometry cell sorting and cloned under limiting dilutionconditions. The obtained clonal populations were used for furtherexperiments.

TABLE I Data obtained for all peptide submixtures % CD4⁺ % CD4⁺ PeptidesIFN-γ⁺ IL-2⁺ None 0.09 0.02 SSX-2 P1-3: SEQ ID NO: 26, 1.17 0.39 SEQ IDNO: 1, SEQ ID NO: 27 SSX-2 P4-6: SEQ ID NO: 28, 0.29 0.05 SEQ ID NO: 29,SEQ ID NO: 30 SSX-2 P7-9: SEQ ID NO: 31, 0.13 0.04 SEQ ID NO: 32, SEQ IDNO: 33 SSX-2 P10-12: SEQ ID NO: 34, 0.09 0.01 SEQ ID NO: 35, SEQ ID NO:36 SSX-2 P13-15: SEQ ID NO: 37, 0.17 0.03 SEQ ID NO: 38, SEQ ID NO: 39

Example 2 Mapping of the Minimal Peptide Optimally Recognized by SSX-2Specific CD4⁺ T Cells

To more precisely define the SSX-2-derived peptide optimally recognizedby specific CD4⁺ T cells from patient LAU 672, we analyzed the relativecapacity of peptide SEQ ID to NO:1 extended or truncated variants tostimulate IFN-γ secretion by specific clonal T cells (clone 2C3). Asillustrated in FIG. 2, both extension and truncation of peptide SEQ IDNO:1 C-terminus resulted in decreased peptide recognition. Truncation ofthe first 6 amino acids at the N-terminus did not significantly affectrecognition (SEQ ID NO:5 and SEQ ID NO:6). In contrast, truncation of 2additional N-terminal amino acids resulted in a 10-fold reduction ofpeptide activity (SEQ ID NO:7). Thus, among analyzed peptides, SEQ IDNO:6 was the minimal peptide optimally recognized by SSX-2 specific CD4⁺T cells, with SEQ ID NO:8 representing the core peptide sequence forpeptides recognized by CD4⁺ T cells. Similar results were obtained usinganother CD4⁺ T cell clone (not shown).

Example 3 SSX-2 13-34 is Recognized by Specific CD4⁺ T Cells in theContext of HLA-DP101 and DP301

To identify the restriction element used by SSX-2 specific CD4⁺ T cells,recognition of peptide SSX-2 13-34 (SEQ ID NO:1) was carried out in thepresence of antibodies that specifically block the recognition ofantigens restricted by different MHC Class II elements (HLA-DR, HLA-DPor HLA-DQ). As illustrated in FIG. 3, anti-HLA-DP antibodies abolishedthe ability of SSX-2 specific CD4⁺ T cells to recognize peptide 13-34(SEQ ID NO:1). In contrast, no significant inhibition was observed usinganti HLA-DR or anti-HLA-DQ antibodies (FIG. 3). Under similarexperimental conditions, no significant inhibition of recognition ofpeptide SSX-2 41-49 by specific CD8⁺ T cells was observed (not shown).To attempt establishing the HLA-DP presenting allele(s) we firstanalyzed the frequency at which PBMC from healthy donors were able topresent the SSX-2 epitope to CD4⁺ T cells. We obtained presentation by 3out of 14 PBMC analyzed suggesting a frequency of the presentingallele(s) in the test population (Caucasian) of about 20% (not shown).Presentation was to further assessed by using a series of HLA-DP typedAPC including autologous APC from patient LAU 672 (Table II). PatientLAU 672 expressed HLA-DP101 and DP402. Peptide presentation in thecontext of the DP402, as well as DP401 (the more frequently expressed DPalleles) was excluded by the lack of presentation obtained with APCexpressing the corresponding alleles. Thus, DP101 was the presentingallele in the case of patient LAU 672. In addition, peptide presentationwas obtained using DP301/DP402 expressing APC indicating that thepeptide can also be recognized by T cells in the context of the DP301allele. The expression frequency of DP101 and DP301 (reported for theFrench population, (Charron D. et al., 1997, In XIIth InternationalHistocompatability Workshop and Conference) is of about 7 and 9%respectively, in good agreement with the frequency of presentingindividuals in the panel of healthy donors analyzed.

TABLE II The ability of APC bearing different HLA-DP alleles to presentpeptide SSX-2 13-34 (SEQ ID NO: 1) was assessed by intracellular IFN-γsecretion SSX-2₁₃₋₃₄ (SEQ ID NO: 1) APC HLA-DP presentation LAU 672/DC101-402 + LAU 149/EBV 301-401 + LAU 465/DC 101-401 + LAU 42/EBV 401-501− LAU 50/TCL 401-402 −

Example 4 The T Cell Epitope Recognized by SSX-2 Specific CD4⁺ T Cellsfrom LAU 672 is not Presented by Tumor Cells but is EfficientlyProcessed and Presented by Professional Antigen Presenting Cells

To assess if the T cell epitope recognized by SSX-2 specific CD4⁺ Tcells from LAU 672 is naturally presented on the surface of tumor cells,we tested different melanoma cells lines for their capacity to presentpeptide SSX-2 13-34 (SEQ ID NO:1) to specific T cell clones. Weidentified two lines that efficiently presented the peptide indicatingexpression of the appropriate restriction elements (FIG. 4). One ofthese lines (Me 260, derived from to patient LAU 149) expressed SSX-2,but was not recognized by CD4⁺ T cells in the absence of exogenouslyadded peptide. Because the expression level of HLA-DP in this line waslow, we treated the cells with IFN-γ for 48 hr. This resulted inincreased expression of HLA-DP (not shown) but not in recognition ofendogenously expressed antigen (FIG. 4). The second melanoma cell line(T465A derived from patient LAU465) able to present peptide SSX-2 13-34to specific CD4⁺ T cells was SSX-2 negative but expressed HLA-A2 andrelatively high levels of HLA-DP (not shown). Transfection of T465Acells with SSX-2 did not result in recognition by specific CD4⁺ T cells.In contrast T465A cells transfected with a plasmid encoding SSX-2 wererecognized by CD8⁺ T cells specific for peptide SSX-2 41-49 (FIG. 4).Similar results were obtained upon IFN-γ treatment. Together theseresults indicate that the T cell epitope recognized by SSX-2 specificCD4⁺ T cells from LAU 672 is not presented by tumor cells at levelssufficient to allow direct recognition of SSX-2 expressing tumors.

We then assessed the ability of professional APC to process the SSX-2antigen and present the SSX-2 13-34 epitope to specific CD4⁺ T cells. Asillustrated in FIG. 5A, EBV cells from patient LAU 149 (EBV LAU 149)were able to efficiently process the SSX-2 protein and present therelevant epitope to the SSX-2 13-34 specific CD4⁺ T cell clone 3C8. Theclone was not significantly stimulated by incubation with the SSX-2protein in the presence of EBV cells unable to present the peptide or byincubation of EBV LAU 149 with NY-ESO-1 protein. Efficient presentationwas also obtained by using LAU 672 autologous dendritic cells (DC) (FIG.5B). Interestingly, both in the case of EBV and DC, processing ofexogenous SSX-2 protein did not result in recognition of the CD8⁺ T cellepitope SSX-2 41-49 by specific clonal CD8⁺ T cells (clone B3.4, FIG.5B).

In this study, we have used a mixture composed of 20-22 amino acid longpeptides spanning the SSX-2 protein sequence and overlapping by 10 aminoacids, to stimulate enriched CD4+ T lymphocytes from an SSX-2 expressingmelanoma patient with autologous irradiated CD4− cells. After a singlein vitro stimulation, peptide submixtures, and then single peptides,were used for screening together with autologous PBMC as a source ofAPC. This simple procedure allowed the identification of the first SSX-2derived CD4+ T cell epitope. Once the reactive peptide in the activemixture was identified, specific CD4+ T cells were isolated using acytokine secretion based cell sorting procedure and cloned by mitogen tostimulation under limiting dilution conditions. All clonal populationsisolated were specific. Specific clonal populations were then used tofurther characterize the identified epitope.

After a single stimulation, about 1% of CD4+ T cells in the culturespecifically secreted IFN-γ in response to antigen stimulation,suggesting that a relatively high frequency of these cells may bepresent in circulating lymphocytes from melanoma patient LAU 672, mostlikely as the result of a spontaneous response to the patient'sautologous SSX-2 expressing tumor. In support of this, no response tothe identified epitope was detected, by using the same methodology, innormal donors expressing the appropriate MHC Class II restrictionelement. It is noteworthy that we have previously found a spontaneousresponse to the CD8+ T cell epitope 41-49 in both circulating and tumorinfiltrating lymphocytes of patient LAU 672. We failed to detect SSX-213-34 specific cells among unstimulated CD4+ T cells from the patient byusing ELISPOT, indicating that, although remarkably elevated after asingle cycle of in vitro stimulation, the frequency of these cells wasbelow ELISPOT detection limits ex vivo. No specific responses to theSSX-2 spanning peptides mix were detected using the same method in thecase of four additional melanoma patients bearing SSX-2 expressingtumors including one (LAU 149) whose APC were able to present the SSX-213-34 epitope to specific CD4+ T cells. This suggests that spontaneousCD4+ T cell responses to SSX-2 could be relatively rare, even amongpatients bearing antigen expressing tumors.

It is noteworthy that a high degree of homology exists between SSX-2 andother SSX family members. In particular, the sequence of the SSX-2 CD4⁺T cell epitope identified here is identical to that of SSX-3 whereas inthe case of other SSX family members (e.g., SSX-1, SSX-4 and SSX-5; seeGure et al., Int. J. Cancer 101 (5), 448-453, 2002) differences ofseveral amino acids are present in this region of the correspondingproteins. For some of the shorter peptides demonstrated herein as havingHLA class II binding activity and other activities, several of thepeptides encoded by other SSX genes are very similar or identical. Theserelated peptides (see Table III; including equivalent fragments as shownelsewhere herein for SSX-2) are believed to be functional variants ofthe SSX-2 peptides presented herein, except for SSX-5 isoform a (SEQ IDNO:43), which varies significantly in amino acid sequence. The analysisof CD8⁺ and CD4⁺ T cell response to other SSX proteins, especially SSX-1and SSX-4, that are, together with SSX-2, the most frequently expressedin tumors, is relevant to the development of generic cancer vaccine.

TABLE III Related SSX family peptides  (amino acid 13-34 regions) Gene/Accession  Amino Acid  SEQ  Location No. Sequence ID NO SSX-2₁₃₋₃₄NM_003147 VGAQIPEKIQKAFDDIAKYFSK 1 (isoform a); NM_175698 (isoform b)SSX-2₁₉₋₃₀ EKIQKAFDDIAK 8 SSX-1₁₃₋₃₄ NM_005635 DDAKASEKRSKAFDDIAT YFSK40 SSX-3₁₃₋₃₄ NM_021014 VGAQIPEKIQKAFDDIAKYFSK 41 (isoform a); NM_175711(isoform b) SSX-4₁₃₋₃₄ NM_005636 DDAQISEKLRKAFDDIAKYFSK 42 (isoform a);NM_175729 (isoform b) SSX-5₁₃₋₃₄ NM_021015 VGSQIPEKMQKHPWRQVC DRGI 43(isoform a) SSX-5₁₃₋₃₄ NM_175723 VGSQIPEKMQKAFDDIAKYFSE 44 (isoform b)SSX-6₁₃₋₃₄ NM_ 173357 DDAKASEKRSKAFDDIAKYFSK 45 SSX-7₁₃₋₃₄ NM_173358AGAQIPEKIQKSFDDIAKYFSK 46 SSX-8₁₃₋₃₄ BK000688 DDDKASEKRSKAFNDIAT YFSK 47SSX-9₁₃₋₃₄ BK000689 AGSQIPEKIQKAFDDIAKYFSK 48 Area corresponding to SEQID NO: 8 is underlined; amino acid differences in this area of SSXpeptides relative to SEQ ID NO: 8 are in bold.

The recognition of the identified SSX-2 epitope by specific CD4+ T cellswas HLA-DP restricted. In support of the importance of DP-restrictedimmune responses, including those against tumors, two tumor antigenderived epitopes (from MAGE-A3 and NY-ESO-1) recognized by CD4+ T cellsin association with the HLA-DP4 molecule have been recently identified(Zeng et al., Proc Natl Acad Sci USA 98:3964-3969, 2001; Schultz et al.,Cancer Res 60:6272-6275, 2000). Recognition of SSX-2 13-34 by CD4+ Tcells from patient LAU 672 was restricted by at least two other HLA-DPmolecules. These molecules, HLA-DP101 and DP301 are, after DP4, the mostprevalent DP alleles. Interestingly, they are characterized togetherwith DP9 by the presence of D at position β86 and may possibly define aseparate supertype of peptide binding specificity.

SSX-2 13-34 specific CD4⁺ T cells from patient LAU 672 failed torecognize SSX-2+ tumor cells expressing the presenting restrictionallele, indicating that the identified epitope was not expressed atlevels sufficient to allow direct recognition of tumor cells even in thecase they express MHC Class II. However, both EBV cells and, even moreefficiently, DC were able to process the native antigen (given to APC inthe form of recombinant SSX-2 protein) and present the relevant epitopeto specific CD4⁺ T cells. MHC-class II restricted recognition ofantigens (including tumor antigens) can follow different presentationpathways. Exogenous proteins captured by the APC by endocytosis reachthe endocytic pathway and, are cleaved by proteases into peptides thatthen associate to MHC Class II molecules. Endogenously produced proteinssuch as secretory or membrane-associated can also follow the exogenousclass II presentation pathway, whereas other endogenous proteins such asmelanosomal proteins contain sequences that directly target them toendosomes. In addition there is growing evidence that otherself-proteins not containing endosomal targeting sequences can also gainaccess to the endogenous class II presentation pathway. The epitopetarget of the spontaneous CD4⁺ T cell response in patient LAU 672required processing and presentation of antigen by autologous DC forCD4⁺ T cell activation to occur. Thus, it is likely that processing andpresentation of tumor-derived SSX-2 antigen by autologous professionalAPC through the exogenous pathway was the mechanism through which thisspontaneous CD4⁺ T cell response to the autologous tumors occurred invivo. CD4⁺ T cells that recognize tumor antigen epitopes expressed bytumor cells have also been previously isolated from healthy donors orcancer patients (Zeng et al., 2001; Manici et al., J Exp Med189:871-876, 1999). Interestingly, in the case of the MAGE-A3 antigenboth CD4+ T cell epitopes that are presented or not by antigenexpressing tumors have been described (Chaux et al., J Exp Med189:767-778, 1999; Schultz et al., 2000; Manici et al., 1999). Thus theidentification of an SSX-2 derived CD4⁺ T cell epitope that is notexpressed by tumor cells does not necessarily imply that this nuclearantigen does not have access to the endogeneous class-II presentationpathway.

Other aspects of the invention will be clear to the skilled artisan andneed not be repeated here. Each reference cited herein is incorporatedby reference in its entirety.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention.

1. An isolated nucleic acid molecule encoding an SSX-2 HLA class II-binding peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.
 2. A composition comprising an isolated nucleic acid molecule encoding an SSX-2 HLA class I-binding peptide and an isolated nucleic acid molecule encoding an SSX-2 HLA class II-binding peptide, wherein the isolated SSX-2 HLA class II-binding peptide consists of an to amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.
 3. A composition comprising one or more of the isolated nucleic acid molecules encoding an SSX-2 HLA class II-binding peptides of claim 1 and an isolated nucleic acid molecule encoding one or more HLA class II molecules.
 4. The isolated nucleic acid molecule of claim 1, wherein the HLA-class II binding peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7.
 5. The isolated nucleic acid molecule of claim 4, wherein the HLA-class II binding peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:5, and SEQ ID NO:6.
 6. The isolated nucleic acid olecule of claim 5, wherein the HLA-class II binding peptide consists of the amino acid sequence set forth as SEQ ID NO:1.
 7. The isolated nucleic acid molecule of claim 1, wherein the HLA class II-binding peptide comprises an endosomal targeting signal that comprises an endosomal targeting portion of human invariant chain Ii. 